Protective group, compound protected by said group and device for grafting the protective group on the compound to protect it

ABSTRACT

The protective group having the following formula (I): 
     
       
         Ar—L—  (I) 
       
     
     wherein 
     Ar represents a substantially planar, fused ring system containing at least 4 aromatic rings, and 
     L represents a group containing at least one carbon atom which is capable of bonding to a group to be protected.

This is a division of application Ser. No. 08/288,771, filed Aug. 11,1994 now U.S. Pat. No. 5,869,605, which is a continuation of applicationSer. No. 08/003,698, filed Jan. 13, 1993, abandoned, which is acontinuation-in-part of application Ser. No. 07/920,579, filed Oct. 30,1992, abandoned, which is a 371 of PCT/FR91/01883 filed Dec. 31, 1991.

BACKGROUND OF THE INVENTION

In organic synthesis, in particular multistep synthesis, thepurification of the products obtained can present more problems than thesynthesis itself. This is particularly true in the case of peptidesynthesis, a synthesis which systematically uses protective groups whichcan also play supplementary roles.

Thus, the need to protect, or activate, a certain function in aminoacids has been used for carrying out biphasic peptide syntheses in orderto facilitate the purification steps. Implicit in these techniques, suchas the Merrifield technique (J. Amer. Chem. Soc., 108, 5242 (1986)),however, is the problem of purification of the synthesized peptideswhich has not been completely resolved. One of the most elegantsolutions to this problem would be to modify the peptide, or any othermolecule requiring protection, by binding it to a solid in a mannerwhich is physically reversible, both in the course of working up andpurification. To date no protective group has been disclosed as beingcapable of exercising such a property.

It is for this reason that one of the main aims of the present inventionis to provide a protective group corresponding to the above criteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 and 2 show the X-ray crystal structure of compound (31) of thepresent invention, 17-hydroxymethyltetrabenzo-(a,c,g,i)fluorene(Tbfmoc), as described in the Examples.

SUMMARY OF THE INVENTION

The present invention relates to a new protective group and its use, inparticular, in peptide synthesis. It relates more particularly to aprotective group facilitating the purification of compounds, especiallypeptides, during or at the end of a synthesis.

In a first embodiment, the present invention is directed to a protectivegroup having the formula (I):

Ar—L—  (I)

wherein

Ar represents a substantially planar fused ring system containing atleast 4 aromatic rings, and

L represents a group containing at least one carbon atom, which iscapable of bonding to a group to be protected.

In a second embodiment, the present invention is directed to a protectedcompound comprising a protective group as described above attached to agroup of a compound to be protected.

In a third embodiment, the present invention is directed to afluorescent label having the formula:

 Ar—L—  (I)

wherein

Ar represents a substantially planar fused ring system containing atleast 4 aromatic rings; and

L represents a group containing at least one carbon atom which iscapable of bonding to a group to be labelled.

The present invention is further directed to a device, which comprises

a) a chamber filled with a graphite material, and

b) a kit for grafting a protective group as described above onto amolecule.

Additionally, the present invention is directed to a process for thesynthesis or separation of a mixture of compounds, comprising the stepsof:

a) protecting at least one group in at least one compound in a mixtureof compounds to be separated with a protective group as described above,and

b) passing the mixture of compounds through a chamber filled with agraphite material.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a protective group having the followingformula (I):

 Ar—L—  (I)

wherein

Ar represents a substantially planar fused ring system containing atleast 4 aromatic rings, and

L represents a group containing at least one carbon atom, which iscapable of bonding to a group to be protected.

Protective groups of this type are disclosed in R. Ramage et al.,Tetrahedron Letters, 33(3), 385-388 (1992), which is herein incorporatedby reference.

The Ar group preferably contains at least 6 aromatic rings and thearomatic rings are preferably hexagonal. Preferably, Ar does not containa heteroatom.

The L group may be, for example, an alkyl group or, in particular, apeptide group. Generally, the L group may comprise any link, or branch,known to those skilled in the art for forming a link between theprotective group and the molecule to be protected. The L group isgenerally selected such that there is no conjugation between the Argroup and the molecule to be protected.

The L group is preferably connected by a carbon atom to the Ar group.Particular preferred Ar—L— groups include those having the formulae (IA)to (IF):

Ar—(CH₂)_(n)—CRR′—  (IA)

Ar—(CH₂)_(n)—C(CY_(m))(R)—  (IB)

 Ar—(CH₂)_(n)—(CF₂)_(m)—C(R)₂—  (IC)

Ar—(CH₂)_(n)—CH══C(R)—CHR′—  (ID)

Ar—(CH₂)_(n)—CH(SO₂R)—  (IE)

Ar—(CH₂)_(n)—SO₂—(CH₂)₂—  (IF)

wherein

R and R′ are each hydrogen, alkyl, aryl, aralkyl or cycloalkyl;

Y is halogen, such as chlorine, bromine or iodine;

n is an integer of from 0 to 5; and

m is an integer of from 1 to 8.

Particularly preferred Ar—L— groups that have the formula (IA) are thosein which the total number of carbon atoms contained in the groups R andR′ is no more than 15. More particularly preferred are those groups inwhich R is hydrogen, R′ is aryl and n is 0.

Additionally, when the function to be protected is NH₂ or an alcohol,precursors of preferred Ar—L— groups include those having formulae (IG),(IH) and (IJ):

Ar—(CHR)_(n)—CO—CHR₁—COOR′  (IG)

Ar—CO—R′  (IH)

 Ar—(CHR)_(n)—CO—CHR₁—CO—R′  (IJ)

wherein

R, R₁ and R′ are each hydrogen, alkyl, aryl, aralkyl or cycloalkyl, and

n is an integer of from 0 to 5.

Preferred Ar—L— groups have the formula Ar—CHR—L′, in which L′ is adirect bond or a group capable of bonding to a group to be protected andR is a hydrogen atom or an alkyl group, preferably having up to 4 carbonatoms. Especially preferred groups of the present invention have theformula (II):

wherein

n is an integer of 0 or 1;

O means that the surrounding ring is aromatic when n is 1 andnon-aromatic when n is 0;

R₁ and R₂, on the one hand, and R₃ and R₄, on the other hand, are chosenso as each to form a fused aromatic ring system together with the carbonatoms to which they are attached;

R and R′ are each an alkyl group or hydrogen, provided that when n is 1,R′ is absent; and

L′ represents a direct bond or a group capable of bonding to a group tobe protected. More particularly preferred Ar—L— groups are those inwhich R is hydrogen.

To rigidify the protective group, it is possible to provide a group R₅to form one or more supplementary aromatic rings, the rings preferablybeing of hexagonal structure and advantageously not containing aheteroatom, to give a protective group of the formula (III):

wherein X represents a bond to R₅ or a hydrogen atom. Additionally, whenn is 0 there is no bond between the central ring and R₅ and when n is 1the bond between one of R₅ and R₂ and R₅ and R₃ is optional butpreferred.

The total number of aromatic rings is advantageously from 4 to 8. Thefree apexes may be substituted by alkyl groups, advantageously having ashort chain (from 1 to 5 carbon atoms). The total number of carbon atomsin the group (not taking account of the link L) is generally from 20 to60, preferably from 25 to 50, carbon atoms.

For ease of synthesis, R₁ and R₂, on the one hand, R₃ and R₄, on theother hand, and R₅ are advantageously chosen to form a system which issymmetrical relative to the central ring (relative to the bisector ofthe intra-ring angle of the carbon carrying the methylene attached tothe link L′). The groups where n is 0, in particular those withoutradical R₅, are more readily accessible.

The chemistry of the groups according to the invention is the same asfor group Fmoc when n is 0 and the same as for the benzyl group in theother cases, for example when n is 1. The chemistry of the group Fmoc iswell known and reference may be made to the following articles as wellas to the general publications on protective groups: Carpino, J. Amer.Chem. Soc. 92, 5748 (1970); J. Org. Chem. 37, 3404 (1972); Synthesis, p.671 (1983); F. Org. Chem. 48, 77 (1983); Int. J. Peptide Protein Res.22, 125 (1983); and Biopolymers, 22, 2157 (1983).

The above groups are easy to obtain by the following sequence ofreactions or equivalents:

The chemistry of the benzyl group is also well-known and it isappropriate to refer to the teaching and specialized publications on thesubject. Reference may also be made to the following articles whichbroke new ground: Int. J. Peptide Protein Res., 27, 358 (1986);Synthesis, p. 303 (1986); J. Org. Chem., 48, 661 and 666 (1983).

The groups of the present invention may be prepared by those methodsdescribed in the above articles and by well-known techniques, such asFreidel-Crafts reactions.

The above articles indicate to those skilled in the art how to graft theprotective groups specified above to the functions to be protected.These groups thus may modify molecules having, for example, an acid,alcohol, thiol, amine or amide function. The molecules protected in thisway can be molecules of any type. Preferably, molecules protected inthis way are amino acids, peptides, nucleosides and nucleotides. Theconstituent amino acids of the peptides may be natural or obtained bysynthesis. Peptides, amino acids, nucleosides and nucleotides may beprotected or substituted on their various functions. Preferred functionsto be protected include —O—P, —S—P, —O—CO—O—P, —O—CO—NA—P, —O—CO—P,—NA—CO—P, and (−)₂N—CO—P, in which A is hydrogen, alkyl, or aryl and Pis the remainder of the molecule having the function to be protected.

Generally, the protective group of the present invention may be removedfrom the protected function by any of the methods known to those havingskill in the art. For example, when the protective group has the formula(IA), the protective group may be removed by acidolysis. Similarly, whenthe protective group has the formula (IB) or (IC), the protective groupmay be removed by treatment with zinc and acetic acid. Likewise, whenthe protective group has the formula (ID), the protective group may beremoved by treating the protected compound with a catalyst comprising aGroup VIII element, such as palladium, complexed with a water-solublecoordinating agent in an aqueous phase. The protective group having theformula (ID) may also be removed by hydrogenation over a suitablecatalyst.

The present invention also provides a protecting compound comprising agroup as defined above attached, via the L or L′ group, to a leavinggroup, such as a group which contains a nitrogen, oxygen, sulphur,selenium, tellurium, silicon or halogen atom. Examples of leaving groupsare halogen atoms (e.g., F, Cl, Br or I), —OH, —COOH, —NH₂, —CONH₂,—SO₃C₆H₄—pCH₃, —SH, —CN or —Si(CH₃)₃ groups.

In any multi-step synthesis, it is important to select a protectinggroup that is both stable to reaction conditions and easily removed,when desired, to provide a final product. The protecting groupsaccording to the present invention are particular suited for use in themulti-step synthesis of peptides and oligonucleotides, due to theirstability to the reaction conditions normally employed in such synthesesand their easy removability at the end of the synthesis. Additionally,the lipophilic nature of the protecting group of the present inventionenables the synthesis of peptides to occur in water-immiscible organicsolvents.

It has also been shown, surprisingly, that molecules modified inaccordance with the protective groups of the present invention areremarkably well-retained on graphite columns or on columns ofgraphitized materials, such as activated charcoal, and this adsorptionis reversible. The strong affinity of the protecting group of thepresent invention means that it is especially suitable for thepurification of peptides. These modified molecules also present anequally good capacity for chiral separation.

The more aromatic rings carried by the group, the better is theadsorption and the more difficult the elution, and vice versa; theoptimum being defined case by case by those skilled in the art. However,4 to 8 aromatic rings, in addition to the central ring, constitute, ingeneral, a satisfactory compromise both from the economic point of viewand from the point of view of the adsorption and elution criteria.

It is expedient to include under the term graphitized materials thosewhich are carbonaceous throughout or at the surface and have undergone agraphitization treatment, in general by pyrolysis. The use of activatedcharcoal provides equivalent binding as with graphite and is preferred,as activated charcoal is cheaper than graphitized carbon (PGC).

The groups according to the present invention are also absorbers ofvisible or ultraviolet radiation in wavelengths which differ from thosefor natural amino acids. Some of these groups fluoresce at wavelengthsdifferent from those of the natural amino acids. Accordingly, the groupsaccording to the present invention may be used as fluorescent labels,attached to a molecule of interest via the L group. For example,17-hydroxytetrabenzo-(a,c,g,i)fluorene (Tbfmoc) can act as a fluorescentlabel, having an excitation wavelength of 383 nm and an emissionwavelength of 424 nm. This is particular advantageous in immunology,where detection of compounds present in very low concentrations requireshighly sensitive techniques.

The Tbfmoc group is particularly suitable for biological applications,such as pharmacology and immunology, in part due to its lack ofmutagenic activity. For example, the Tbfmoc group did not exhibitmutagenic activity in strains of Salmonella typhimurium, with andwithout metabolic activation.

The property of absorbance also enables devices to be developedcomprising, for successive or simultaneous use:

a) a chamber, such as a column, filled with a graphite material,preferably graphite or activated charcoal, and

b) a kit for grafting a protective group as defined above on a molecule.

The graphite or graphitized material has been defined above, while thekit for grafting the protective group comprises the various reagentsknown to those skilled in the art for grafting a protective group on amolecule to be protected. This device is advantageously completed by (c)a fraction-collecting system fitted with an ultraviolet (UV), visiblespectroscopic, or fluorescent spectroscopic detector.

Thus, it has been possible to develop a new process for synthesis and/orseparation of molecules, in particular peptide fragments, whichcomprises the following steps:

protecting at least one group in at least one compound in a mixture ofcompounds to be separated with a group as defined above, and

passing the mixture of compounds through a chamber filled with agraphite material.

The present invention is now further illustrated in the followingExamples:

All amino acid derivatives were purchased from either Fluka, Aldrich orSigma. Melting points were taken in open capillaries on an electricallyheated Buchi 510 melting point apparatus, or on microscope slides on anelectrically heated Reichert 7905 hot stage and are uncorrected.Thin-layer chromatography (t.l.c.) was carried out using plastic sheetscoated with silica gel 60 GF-254 (Merck 5735) in the following systems:

(A) ethyl acetate/petrol (b.p. 40-60° C.) (1:4)

(B) methanol/chloroform (1:9)

(C) toluene

(D) chloroform

(E) ethyl acetate

(F) benzene

(G) dichloromethane

(H) methanol/chloroform/acetic acid (1:9:0.5)

(I) n-butanol/acetic acid/water (3:1:1)

Visualization of the compounds was achieved by a suitable combination ofthe following methods: iodine vapor, ultraviolet absorption at 254 nm or352 nm, neutral potassium permanganate and bromophenol blue sprays,Mary's reagent (4,4′-bis(dimethylamino)diphenyl carbinol) for acidfunctions and ninhydrin for compounds with free amino groups. Opticalrotations were measured on an AA 1000 polarimeter using a 1 dm cell inthe solvents quoted in the text. High performance liquid chromatography(HPLC) was performed using either a Waters system, i.e., 2×600 A pumps,a U6K injector, a 680 automatic gradient controller, a model 441ultraviolet detector; or an Applied Biosystems system, i.e., 2×1406 Asolvent delivery system, a 1480 A injector/mixer, and a 1783 Adetector/controller. Analytical separations were carried out on aWhatman Partisil-5 ODS-3 (4.6 mm ID×250 mm) and on a ABI (RP18) aquaporeOD-300, 300 Å pore size, 7 μm spherical silica (4.6 mm ID×220 mm)column, under the conditions indicated in the text. Flash chromatographywas performed using silica gel 60 (230-400 mesh (Fluka); 60-100 g ofsilica per gram of crude material; active length 15-20 cm). Infraredspectra were recorded on a Perkin Elmer 781 spectrophotometer in thesolvent indicated or by the KBr disc technique, using polystyrene as thestandard (1603 cm⁻¹). Ultraviolet spectra were recorded on a Cary 210spectrophotometer and fluorescence spectra on a Perkin Elmer LS-5luminescence spectrophotometer. Mass spectra were measured on a KratosMS 50 TC machine. Proton nuclear magnetic resonance (NMR) spectra wererecorded on either Bruker WP 80 (80 MHz), WP 200 (200 MHz) or WH 360(360 MHz) machines in the solvent indicated, using externaltetramethylsilane as the standard (δ=0.00). Carbon-13 NMR spectra wererecorded on a Bruker WP 200 (50 MHz) or WH 360 (90 MHz) machines in thesolvent indicated, using tetramethylsilane as the standard. Elementalanalyses were carried out on a Carlo Erba model 1106 elemental analyzer.Single crystal X-ray structure determination was performed on a StoeStadi-4 four-circle diffractometer, graphite-monochromated (Cu-Kαradiation, λ=1.54184 Å). All solvents were distilled before use and thefollowing were dried using the reagents given in parentheses whenrequired: benzene (sodium wire), chloroform (phosphorus pentoxide),dichloromethane (calcium hydride), diethyl ether (sodium wire), dioxan(neutral alumina and sodium wire), methanol (magnesium-iodine), toluene(sodium wire). Petrol (b.p. 40-60° C.) refers to that fraction whichboils between 40° C. and 60° C.

EXAMPLE 1 Synthesis of 13-hydroxymethyldibenzo(a,c)fluorene

Methyl (phenanthren-9-yl, phenyl)hydroxyacetate (25)

The synthesis of methyl(phenanthren-9-yl, phenyl) 5 hydroxyacetate (25)was carried out in ether by reaction of 9-phenanthrenyl lithium (23)with methyl benzoylformate.

To a cold (0° C.) solution of 9-bromophenanthrene (2.57 g, 10 mmol) indry ether (10 ml) under nitrogen, was added a solution of n-butyllithiumin hexane (8 ml, 11 mmol; 1.1 equiv; 1.38M titrated) dropwise over 10min. The reaction mixture was stirred for one hour at room temperature.The resultant suspension was allowed to settle and then the supernatantremoved by syringe. The residue was resuspended in ether (5 ml). Thissolution was added, under nitrogen, to a cold (0° C.) solution of methylbenzoylformate (1.64 g, 1.4 ml, 10 mmol) in 50 ml of diethylether. Theresulting mixture was heated under reflux for 2 h and allowed to stirovernight. After addition of dilute (10% v/v) HCl (50 ml; pH=1), theorganic layer was separated, combined with ether washings (2×50 ml) ofthe aqueous layer, washed with water (2×50 ml) and dried with MgSO₄. Thesolvent was removed under vacuum to give a yellow oil. Afterpurification by flash chromatography (eluent: (A)), a yellow oil wasobtained which was triturated with petrol. A white solid was obtainedwhich was filtered and dried in a vacuum desiccator to give compound(25) (1.75 g, 51%); m.p. 143-145° C. (Found: C, 79.7; H, 5.24; C₂₃H₁₈O₃requires: C, 80.7; H, 5.26%); t.l.c. R_(f)(A) 0.32; R_(f)(B) 0.76;ν_(max) CH₂Cl₂ 3690 (free OH), 3510 (H bonded OH), 3025 (CH stretching,aryl), 2960, 2880, 2860 (CH stretching, alkyl), 1730 (CO, ester), 1600,1500 (aromatic rings), 1225, 1185, 1170, 1110, 1100, 1070 (COstretching) cm⁻¹; λ_(max) 296 (9832), 284 (9200), 276 (11981), 254(48450), 248 (37620) nm; δH (CDCl₃, 200 MHz) 8.72 (1H, d, ³J=8 Hz,aromatic), 8.66 (1H d, ³J=8 Hz, aromatic), 8.16 (1H, d, ³J=8 Hz,aromatic), 7.75-7.25 (11H, m, aromatic), 4.32 (1H, s, OH), 3.85 (3H, s,CH₃); δC (CDCl₃, 50 MHz) 175.7 (CO, ester), 141.1, 135.6, 131.3, 130.6,130.3, 129.8 (quaternary aromatic C's), 129.1, 128.2, 128.1, 127.3,127.0, 126.9, 126.6, 126.2, 126.1, 122.9, 122.2 (aromatic CH's), 82.1(C₁, quaternary), 53.4 (CH₃); m/z (EI) 342, 283, 105.

13-Carboxymethyldibenzo(a,c)fluorene (26)

Treatment of (25) under acidic conditions (conc. H₂SO₄, CH₃COOH at 10°C.) led to the formation of (26) in poor yield.

Surprisingly, treatment of (25) with PPA at 110° C., led to theformation of the desired ester (26) in 42% yield.

Polyphosphoric acid (60 g) was heated to 110° C. stirred with amechanical stirring paddle. To this was addedmethyl(phenanthren-9-yl,phenyl)hydroxyacetate (5 g, 14.6 mmol). Themixture was stirred for 1 hour at 110° C. The reaction was then cooledto room temperature, diluted with water (150 ml) and extracted withethyl acetate (4×250 ml). The organic layers were combined, washed withNaHCO₃ (10% v/v; 200 ml), water (100 ml) and dried over MgSO₄. Thesolvent was removed in vacuo to give a yellow solid. This crude solidwas purified by flash chromatography using toluene as the eluent. Afterchromatography compound (26) (2.8 g, 60%) could be isolated. This solidwas recrystallized from CH₂Cl₂/petrol (b.p. 40-60° C.) to afford a whitesolid, (1.96 g, 42%); m.p. 190-191° C.; (Found: C, 85.2; H, 4.93;C₂₃H₁₆O₂ requires: C, 85.2; H, 4.94%); t.l.c. R_(f)(C) 0.50; R_(F)(D)0.56; ν_(max) (CH₂Cl₂), 3040, 3020 (CH stretching, aryl), 2970 (CHstretching, alkyl), 1730 (CO, ester), 1610, 1600, 1580 (aromatic rings)cm⁻¹; λ_(max) 364 (540), 346 (16200), 322 (18360), 268 (64800) nm; δH(CDCl₃, 200 MHz), 8.84-8.67 (3H, m, aromatic), 8.36 (1H, d, ³J=7.8 Hz,aromatic), 7.98-7.90 (1H, m, aromatic), 7.80-7.35 (7H, m, aromatic),5.16 (1H, s, CH), 3.63 (3H, s, CH₃); δC (CDCl₃, 50 MHz), 172.0 (CO,ester), 142.7, 141.9, 137.4, 135.6, 131.1, 130.0, 128.7, 128.4(quaternary aromatic C's), 128.1, 127.0, 126.7, 126.3, 126.2, 124.3,124.1, 123.8, 123.3, 123.2, 122.9 (aromatic CH's), 53.4 (CH₃), 52.4(CH); m/z (EI) 324, 265, 262, 132.

13-Hydroxymethyldibenzo(a,c)fluorene (21)

The reduction of ester (26) was achieved in 48% yield using threeequivalents of diiobutylaluminum hydride (DIBAL-H) in dichloromethane at−50° C.

Diisobutylaluminum hydride (4.6 ml, 4.62 mmol; 1 M in CH₂Cl₂) was addedat −65° C. to a solution of 13-carboxymethyl-dibenzo(a,c)fluorene (0.5g, 1.54 mmol) in dichloromethane (10 ml). The reaction mixture wasstirred for 3 h and the temperature maintained between −50° C. and −40°C. A white precipitate was obtained. The reaction mixture was treatedwith a mixture of acetic acid and water (1:1; 30 ml) and extracted withdichloromethane (3×60 ml). The organic layer was washed with saturatedbicarbonate (50 ml), water (30 ml) and dried over MgSO₄. The solvent wasremoved in vacuo to afford a crude solid. This crude solid was purifiedby flash chromatography using chloroform as the eluent. Compound (21)was obtained as a white solid which was washed with petrol (0.217 g,48%); m.p. 167-168° C.; (Found: C, 89.0; H, 5.39; C₂₂H₁₆O requires: C,89.2; H, 5.41%); t.l.c. R_(f)(D) 0.26; R_(f)(E) 0.64; ν_(max) (CH₂Cl₂)3610 (free OH), 3490 (H bonded OH), 3060 (CH stretching), 2940, 2890 (CHstretching, alkyl), 1610, 1600, 1580 (aromatic rings) cm⁻¹; λ_(max) 366(888), 338 (12728), 322 (14504), 266 (27232), 246 (31968) nm; δH (CDCl₃,200 MHz), 8.88-8.68 (3H, m, aromatic), 8.38 (1H, d, ³J=7.9 Hz,aromatic), 8.14-8.08 (1H, m, aromatic), 7.81-7.33 (7H, m, aromatic),4.45 (2H, m, H_(a) CH₂ and H_(c)), 3.85 (1H, m, H_(b), CH₂), 1.64 (1H,s, OH), δC (CDCl₃, 50 MHz), 146.7, 142.5, 139.4, 134.9, 130.9, 130.1,128.7 (quaternary aromatic C's), 127.5, 126.8, 126.1, 125.9, 125.8,124.7, 124.2, 124.1, 123.8, 123.4, 122.9 (aromatic CH's), 65.7 (CH),50.0 (CH); m/z (EI) 296, 265.

This route provides a synthesis of 13-hydroxymethyl-dibenzo(a,c)fluorene(21) in four steps with an overall yield of 10%.

The acetate of both the alcohol (21) and 9-fluorenemethanol(preparations shown below) were then prepared in order to compare theirbehavior on an HPLC column packed with PGC.

9-Fluorenylmethyl acetate (29) gave a retention time of 3.3 min. (elutedwith CHCl₃), whereas 13-acetoxymethyl dibenzo(a,c)fluorene (30) underthe same conditions, was not completely retained on the column but wasslowly eluted.

Preparation of 13-Acetoxymethyldibenzo(a,c)fluorene (30)

13-Hydroxymethyldibenzo(a,c)fluorene (215.2 mg, 0.727 mmol) wasdissolved in acetic anhydride (5 ml). To this solution was added onedrop of sulfuric acid (2M). The reaction mixture was stirred for 30 min.A white precipitate was obtained which was filtered and washed withwater (150 ml). The solid was finally dried to constant weight over P₂O₅in a vacuum desiccator to afford 13-acetoxymethyldibenzo(a,c)fluorene asa white solid, (206.3 mg, 84%); m.p. 135-136° C.; (Found: C, 84.9; H,5,33; C₂₄H₁₈O₂ requires: C, 85.2; H, 5.33%; t.l.c. R_(f)(D) 0.57;R_(f)(E) 0.68; ν_(max) (CH₂C₂) 3030 (CH stretching, aryl) 2980, 2920 (CHstretching, alkyl) 1735 (CO, ester), 1610, 1600, 1570 (aromatic rings)cm⁻¹; λ_(max) 366 (1184), 338 (25160), 322 (27528), 265 (52688), 246(53020) nm; δH (CDCl₃, 200 MHz), 8.86-8.69 (3H, m aromatic), 9.38 (1H,d, ³J=7.9 Hz, aromatic), 8.30-8.25 (1H, m, aromatic), 7.78-7.35 (7H, m,aromatic), 5.16 (1H, dxd, ³J_(a,c)=4.1 Hz, ²J_(a,b)=10.8 Hz, H_(a),CH₂), 4.56 (1H, dxd, ³J_(c,a)=4.1 Hz, ³J_(c,b)=9.3 Hz, Hc), 3.76 (1H,dxd, ²J_(b,a)=10.8 Hz, ³J_(b,c)=9.3 Hz, Hb, CH₂), 2.17 (3H, s, CH₃);δ_(C) (CDCl₃, 50 MHz) 171.0 (CO, ester), 146.7, 142.1, 138.7, 134.8131.1, 130.1, 128.7, 128.6 (quaternary aromatic C's), 127.6, 127.0,126.8, 126.2, 126.1, 125.7, 124.9, 124.8, 124.3, 123.4, 123.3, 122.9(aromatic CH's), 67.3 (CH₂), 46.5 (CH), 20.9 (CH₃); m/z (EI) 338, 277,265, 139, 43.

HPLC: column (ODS3-PL5-393, solvents: A(H₂O), B(CH₃CN),

conditions: B(50%) 2 min.

λ=254 nm, flow rate: 1 ml/min., injection: 5 μl,

C=1.42 mg/ml, AUFS=2, retention time: 21.6 min.

Preparation of 9-Fluorenylmethylacetate (29)

9-Fluorenylmethanol (0.215 g, 1.1 mmol) was dissolved in aceticanhydride (5 ml). To this solution was added 2 drops of sulfuric acid(2M). This was stirred for 30 min. The clear solution was poured ontocold water (20 ml). The precipitate obtained was collected on a Buchnerfilter and dried to constant weight over P₂O₅ in a vacuum desiccator toafford 9-fluorenyl-methylacetate as a white solid, (0.175 g, 67%); m.p.83-84° C.; (Found: C, 80.5; H, 5.98; C₁₆H₁₄O₂ requires: C, 80.7; H,5.88%); t.l.c. R_(f) (D) 0.50; R_(f) (E) 0.68; ν_(max) (CH₂Cl₂) 3030 (CHstretching), 2960, 2910 (CH stretching, alkyl), 1735 (CO, ester), 1610(aromatic rings) cm⁻¹; λ_(max) 300 (2476), 290 (2063), 268 (7634) nm;δ_(H) (CDCl₃, 80 MHz), 7.83-7.29 (8H, m, aromatic), 4.46-4.28 (3H, m,CH, CH₂), 2.14 (3H, s, CH₃); δ_(C) (CDCl₃, 50 MHz), 170.8 (CO, ester),143.6, 141.1 (quaternary aromatic C's), 127.6, 126.9, 124.9, 119.9(aromatic CH's), 66.2 (CH₂), 46.5 (CH), 20.8 (CH₃); m/z (EI) 238, 178,165, 149, 60, 43.

HPLC: column (ODS3-PL5-393), solvents: A(H₂O), B(CH₃CN)

Conditions: B(50%) 2 min.

λ=254 nm, flow rate: 1 ml/min.,

injection: 5 μl, C=1.46 mg/ml, AUFS=2, retention time: 8.4 min.

EXAMPLE 2 Synthesis of 17-Hydroxymethyltetrabenzo(a,c,g,i)fluorene (31)

The tetrabenzofluorenyl alcohol (31) was prepared follows:

(a) BuLi, Et₂O, r.t., 30 min.; EtOOCCOOEt, Et₂O, 0° C.-5° C., 2 h, 63%;

(b) 9-Bromophenanthrene, BuLi, Et₂O, r.t., 30 min.; (32), EtO, 0° C.-5°C., 2 h, 46%; (c) PPA, 140° C., 4 h, 49%; (d) DIBAL-H (3 equiv.),CH₂Cl₂, −65° C., 1 h, 73%.

Ethyl,2-oxo-2-(phenanthren-9′-yl)acetate (32)

The α-keto ester (32) was synthesized by adding 9-phenanthrenyl lithiumto a solution of diethyl oxalate (20% excess) in ether at 0° C. Thereaction mixture was subsequently heated under reflux for 2 hours.Following purification by flash chromatography, the product was isolatedin 23% yield.

The α-keto ester (32) can also advantageously be prepared in ether, bycarefully adding 9-phenanthrenyl lithium to a solution of diethyloxalate (20% excess), between 0° C. and 5° C., and subsequently stirringthe reaction at room temperature.

To a stirred solution of 9-bromophenanthrene (5.14 g, 20 mmol) in dryether (20 ml) was added at 0° C., under nitrogen, a solution ofn-butyllithium in hexane (16 ml, 22 mmol; 1.1 equiv; 1.38M titrated)dropwise over 10 min. The reaction was stirred for 1 h at roomtemperature. The resultant suspension was allowed to settle and thesupernatant was removed by syringe. The residue was resuspended in ether(10 ml). This suspension was added, under nitrogen, to a cold (0° C.)solution of diethyl oxalate (3.4 ml, 25 mmol) in ether (100 ml). Thetemperature was maintained between 0° C. and 5° C. during the addition.The reaction mixture was then stirred for 2 h between 0° C. and 5° C.and finally at room temperature for 2 h. After addition of dilute HCl(100 ml; 10% v/v), the organic layer was separated, combined with ethylacetate washings (3×100 ml) of the aqueous layer, neutralized withNaHCO₃ solution (100 ml; 1M), washed with water (50 ml) and dried overMgSO₄. The solvent was removed in vacuo to give an orange oil which wastriturated with petrol (b.p. 40° C.-60° C.). Compound (32) was obtainedas a yellow solid which was filtered and dried, (3.53 g 63%) m.p. 67-68°C.; (Found: C, 77.5; 5.02; C₁₈H₁₄O₃ requires: C, 77.7; H, 5.04%); t.l.c.R_(f) (A) 0.56, R_(f) (D) 0.52; ν_(max) (CH₂Cl₂) 3070 (CH stretching,aromatic), 2990, 1910, 2880 (CH stretching, alkyl), 1735 (CO, ester),1680 (CO, ketone), 1620, 1575 (aromatic rings) cm⁻¹; λ_(max) 328 (982),286 (7784), 252 (34842) nm; δ_(H) (CDCl₃, 200 MHz) 9.04 (1H, m,aromatic), 8.6 (2H, m, aromatic), 8.23, (1H, s, H₁₀ aromatic), 7.92 (1H,d, ³J=7.6 Hz, aromatic), 7.78-7.59 (4H, m, aromatic), 4.51 (2H, 9,³J=7.1 Hz, CH₂), 1.48 (3H, t, ³J=7.1 Hz, CH₃), δ_(C) (CDCl₃, 50 MHz)188.5 (CO, ketone), 164.4 (CO, ester), 137.2 (aromatic CH), 132.8(quaternary aromatic), 130.5, 130.2 aromatic CH's), 129.2, 128.1(quaternary aromatic C's), 128.0, 127.4, 127.1, 126.3, 122.7, 122.6(aromatic CH's), 62.3 (CH₂), 14.0 (CH₃); m/z (EI) 278, 205, 177, 176.

Ethyl(bis-phenanthren-9′-yl)hydroxy acetate (33)

The tertiary alcohol (33) was synthesized in adequate yield undersimilar conditions using (32) as the keto ester component.

To a stirred solution of 9-bromophenanthrene (2.57 g, 10 mmol) in dryether (10 ml) was added, at 0° C. under nitrogen, a solution ofn-butyllithium in hexane (8 ml, 11 mmol; 1.1 equiv.; 1.38 M titrated)dropwise over 10 min. The reaction mixture was stirred for 1 h at roomtemperature. This solution was added, under nitrogen, to a cold (0° C.)solution of ethyl,2-oxo-2-(phenanthren-9′-yl)acetate (2.78 g, 10 mmol)in ether (50 ml). The temperature was maintained between 0° C. and 5° C.during the addition. The reaction mixture was finally stirred at roomtemperature for 2 h. After addition of dilute HCl (50 ml; 10% v/v) theorganic layer was separated, combined with ethyl acetate washings (3×250ml) of the aqueous layer, neutralized with a solution of NaHCO₃ (1M; 200ml), washed with water (200 ml) and finally dried over MgSO₄. Thesolvent was removed in vacuo to give a residue. After purification byflash chromatography using chloroform/petrol (b.p. 40° C.-60° C.) (4:1),the expected product was obtained from the fractions containing materialof R_(f)=0.23. After recrystallization from dichloromethane/petrol (b.p.40° C.-60° C.), a white solid was obtained which was filtered and driedto give compound (33) (2.11 g, 46%); m.p. 188-189° C.; (Found: C, 83.1;H, 5.23; C₃₂H₂₄O₃ requires: C, 84.2; H, 5.26%); t.l.c. R_(f) (E) 0.71;R_(f) (D) 0.48; ν_(max) (CH₂Cl₂) 3680 (free OH), 3510 (H bonded OH),3060 (CH stretching, aromatic), 2990, 2940 (CH stretching, alkyl), 1735(CO, ester), 1600 (aromatic ring) cm⁻¹; λ_(max) 332 (629), 300 (25080),288 (23560), 256 (123120) nm; δ_(H) (CDCl₃, 200 MHz), 8.80-8.69 (4H, m,aromatic), 8.51 (1H, s, H₁₀, aromatic), 8.47 (1H, s, H₁₀, aromatic),7.72-7.41 (12H, m, aromatic), 4.46 (1H, s, OH), 4.41 (2H, 9, ³J=7.1 Hz,CH₂), 1.17 (3H, t, ³J=7.1 Hz, CH₃), δ_(C) (CDCl₃, 50 MHz) 175.7 (CO,ester), 135.0, 131.4, 130.6, 130.5, 130.1 (quaternary aromatic C's),129.2, 128.2, 127.3, 126.6, 126.2, 126.1, 122.9, 122.3 (aromatic CH's),84.3 (C₁, quaternary), 62.9 (CH₂), 13.8 (CH₃); m/z (EI) 383, 206, 177,176.

17-Carboxyethyltetrabenzo(a,c,g,i)fluorene (34)

Treatment of (33) with polyphosphoric acid at 140° C. led to theformation of the cyclic ester (34) in 49% yield via4π-electrocyclization of the α-ethoxycarbonyl diaryl cation

Purification of (34) is difficult due to the formation of side products.Recrystallization of the crude material following flash chromatographycan be carried out.

Polyphosphoric acid (20 g) was placed in a three necked 100 ml roundbottom flask equipped with a mechanical stirring paddle. Ethyl(bis-phenanthren-9′-yl) hydroxy acetate (0.402 mg, 0.881 mmol) was thenadded and the reaction was stirred at 140° C. for 4 h and subsequentlyat room temperature overnight. The reaction mixture was diluted withwater (50 ml) and extracted with ethyl acetate (4×50 ml). The organiclayer was neutralized with NaHCO₃ (1M; 2×30 ml), washed with water (30ml) and dried over MgSO₄. After removal of the solvent in vacuo aresidue was obtained which was purified by flash chromatography (eluent:benzene). The fractions containing material of R_(f)=0.68 wereevaporated to give compound (34) as a yellow solid which was washed withpetrol, filtered and dried, (189 mg, 49%); m.p. 165-166° C.; (Found: C,84.4; H, 4.66; C₃₂H₂₂O₂ requires: C, 87.7; H, 5.02%); t.l.c. R_(f) (D)0.68, R_(f) (F) 0.62; ν_(max) (CH₂Cl₂) 3060 (CH stretching, aromatic),2990, 2960, 2920, 2860 (CH stretching, alkyl), 1810 (CO, lactone), 1735(CO, ester), 1610, 1500 (aromatic rings) cm⁻¹; λ_(max) 386 (6371), 370(7964), 302 (22697), 290 (10894), 254 (42208) nm; δ_(H) (CDCl₃, 200 MHz)8.78-8.61 (6H, m, aromatic), 8.28-8.21 (2H, m, aromatic), 7.75-7.55 (8H,m, aromatic), 5.39 (1H, s, CH), 4.04 (2H, q, ³J=7.1 Hz, CH), 0.97 (3H,t, ³J=7.1 Hz, CH₃); δ_(C) (CDCl₃, 67 MHz) 171.9 (CO, ester), 138.3,138.2, 131.6, 130.5, 128.6, 127.7 (quaternary aromatic C's), 127.6,127.1, 126.2, 126.1, 125.0, 123.9, 123.4, 123.2 (aromatic CH's), 61.4(CH₂), 54.9 (CH), 13.8 (CH₃); m/z (EI), 438 (M⁺).

17-Hydroxymethyltetrabenzo(a,c,g,i)fluorene (31)

Reduction of ester (34) to the corresponding alcohol (31) proceeded in astraightforward manner (73% yield). The reaction was carried out in DCMat −65° C. using 3 equivalents of DIBAL-H.

Following flash chromatography and recrystallization from DCM, crystalsof (31) were obtained and proved suitable for X-ray analysis.

Diisobutylaluminum hydride (12.3 ml, 12.3 mmol; 3 equiv.; 1M solution inCH₂Cl₂) was added dropwise at −65° C. to a solution of17-carboxyethyltetrabenzo(a,c,g,i) fluorene (1.795 g, 4.098 mmol) in drydistilled dichloromethane (25 ml). The temperature was maintainedbetween −65° C. and −60° C. during the addition. The reaction mixturewas stirred for 1 h at −65° C. and at room temperature for a furtherhour. The reaction mixture was cooled to −30° C. An aqueous solution ofacetic acid (50 ml; 10% v/v) was then added dropwise. After separationof the two layers, the aqueous phase was extracted with dichloromethane(3×100 ml). The combined organic phases were washed with water (70 ml)and neutralized with NaHCO₃. Finally the organic phase was dried overMgSO₄ and evaporated to give a crude oil (1.7 g). After purification byflash chromatography using benzene as the eluent, the fractionscontaining material of R_(f)=0.14 were evaporated to give a yellowsolid. This was recrystallized from CH₂Cl₃/petrol (b.p. 40° C.-60° C.)to afford compound (31) as a yellow solid (1.18 g, 73%); m.p. 202-203°C.; (Found: C, 91.2; H, 4.96; C₃₀OH₂₀O requires: C, 90.9; H, 5.05%);t.l.c. R_(f) (F) 0.15, R_(f) (A) 0.18, R_(f) (B) 0.75; ν_(max) (CH2Cl₂)3600 (free OH), 3060 (CH stretching, aromatic), 2940, 2900 (CHstretching, alkyl), 1610, 1500 (aromatic rings), 1045 (CO stretching)cm⁻¹; λ_(max) 380 (10692), 368 (11484), 302 (27324), 290 (21780), 54(43560) nm; δ_(H) (CDCl₃, 200 MHz), 8.80-8.63 (6H, m, aromatic),8.27-8.22 (2H, m, aromatic), 7.73-7.56 (8H, m, aromatic), 5.05 (1H, t,₃J=4.3 Hz, CH), 4.49 (2H, d, ³J=4.3 Hz, CH), 1.31 (1H, s, OH); δ_(C)(CDCl₃, 50 MHz), 41.6, 137.3, 131.4, 130.4, 128.5, 127.9 (quaternaryaromatic C's), 127.4, 126.9, 126.1, 125.9, 125.0, 124.5, 123.4 (aromaticCH's), 66.5 (CH₂), 50.8 (CH); m/z (EI) 396 (M⁺), 366.

The crystal structure of the alcohol (31) indicates that there are twomolecules held together by hydrogen bonding between the two hydroxylgroups. FIGS. 1 and 2 show the crystal structure of the alcohol (31).The exocyclic hydroxymethyl group has two different conformations,probably as this is required for the formation of the hydrogen-bondeddimer. The interaction between H₈ and H₉ (d=2.0 Å, Van der Waal's radiusfor hydrogen=1.2 Å) is responsible for a degree of non-planarity in themolecule. However, the molecule is sufficiently planar for the purposesof the invention.

This molecule is chiral if it adopts a fixed orientation, as in thecrystal structure. Therefore it is possible that in solution at lowtemperature, the molecule may also adopt a fixed orientation and bechiral. ¹H n.m.r. at room temperature in CDCl₃ showed a triplet (δ=5.05ppm) as well as a doublet (δ=4.5 ppm), corresponding to H(17) and thetwo protons of the methylene group respectively. A probable explanationis that the phenanthrene rings can oscillate rapidly during theacquisition period and consequently the protons of the CH₂ group aremagnetically equivalent (as are the matched pairs of aromatic protons,e.g., H(2) and H(15)).

Nuclear Overhauser experiments carried out on this compound showed theclose through-space interactions between the methylene group and the twoaromatic protons H(1) and H(16).

EXAMPLE 3 Synthesis of 17-acetoxymethyltetrabenzo(a,c,g,i)fluorene (35)

17-acetoxymethyltetrabenzo(a,c,g,i)fluorene (35) was easily prepared in80% yield from alcohol (31) using a large excess of acetic anhydride anda catalytic amount of sulfuric acid.

As expected, the acetate (35) was totally retained when passed throughan HPLC column packed with PGC (eluent CHCl₃)

17-Hydroxymethyltetrabenzo(a,c,g,i)fluorene (0.207 g, 0.522 mmol) wasdissolved in acetic anhydride (4 ml). To this solution was added 2 dropsof H₂SO₄ (2M). The reaction mixture was stirred for 1 h. Compound (35)was obtained as a yellow solid which was washed with water (80 ml) anddried to constant weight over P₂O₅ in a vacuum desiccator (183.8 mg,80%); m.p. 209-210°, (Found: C, 87.0; H, 5.08; C₃₂H₂₂O₂ requires: C,87.7; H, 5.02%); t.l.c. R_(f) (A) 0.32, R_(f) (F) 0.22; ν_(max) (CH₂Cl₂)3060 (CH stretching, aromatic), 1740 (CO, ester), 1610, 1500 (aromaticrings), 1230, 1045 (CO, stretching) cm⁻¹; λ_(max) 380 (17885), 368(19345), 302 (45260), 290 (36865), 254 (72271) nm; δ_(H) (CDCl₃, 80MHz), 8.83-8.57 (6H, m, aromatic), 8.29-8.17 (2H, m, aromatic),7.78-7.54 (8H, m, aromatic), 5.12 (1H, t, ³J=5.5 Hz, CH), 4.60 (2H, d,³J=5.5 Hz, CH₂), 1.84 (3H, s, CH₃); δ_(C) (CDCl₃, 50 MHz) 170.7 (CO,ester), 141.8, 137.0, 131.5, 130.4, 128.6, 128.0 (quaternary aromaticC's), 127.4, 126.8, 126.1, 126.0, 125.0, 124.9, 123.5, 123.3 (aromaticCH's), 67.9 (CH₂), 47.1 (CH), 20.7 (CH₃); m/z (EI) 438 (M⁺), 378, 364.

EXAMPLE 4 Conversion of 17-hydroxymethyltetrabenzo(a,c,g,i )fluorene tothe chloroformate (37)

The chloroformate (37) is synthesized by the following sequence ofreactions: treatment of alcohol (31) with N,N′-bis-trimethylsilyl urea(2.5 equiv) to give the corresponding trimethylsilyl ether (39),followed by reaction of the latter with phosgene. Followingrecrystallization, the pure product can be obtained in 21% yield.

(a) N,N′-bis-trimethylsilyl urea (2.5 equiv), CH₂Cl₂, reflux, 3 h, 81%;(b) phosgene (7.5 equiv), CH₂Cl₂, reflux, 2 h, 21%.

17-(Trimethylsilyloxymethyltetrabenzo(a,c,g,i)fluorene (39)

A solution of 17-hydroxymethyltetrabenzo(a,c,g,i) fluorene (0.05 g,0.126 mmol) and N,N′-bis-trimethylsilyl urea (32.3 mg, 0.158 mmol; 2.6equiv.) in dichloromethane (2 ml) was heated under reflux for 3 h. Theprecipitated urea was filtered off and washed with dichloromethane (2×1ml). The solvent was removed in vacuo to give a residue. Purification bywet flash chromatography (benzene/petrol (b.p. 40-60° C.) (75:25)) gavecompound (39) (47.8 mg, 81%); m.p. 130-131° C.; (Found: C, 84.2; H,6.06; C₃₃H₂₈OSi requires: C, 84.6; H, 5.98%); t.l.c. R_(f) (F) 0.58,R_(f) (A) 0.51, R_(f) (G) 0.76; δ_(H) (CDCl₃, 80 MHz) 8.86-8.62 (6H, m,aromatic), 8.49-8.37 (2H, m, aromatic), 7.79-7.51 (8H, m, aromatic),5.11 (1H, t, ³J=5.3 Hz, CH), 4.13 (2H, d, ³J=5.3 Hz, CH₂), 0.4 (9H, s,3×CH₃); δ_(c) (CDCl₃, 67 MHz) 143.4, 136.5, 131.4, 130.3, 129.0, 128.1(quaternary aromatic C's), 127.4, 126.5, 125.9, 125.8, 125.6, 124.9,123.5, 123.0 (aromatic CH's), 67.2 (CH₂), 51.4 (CH), −1.0 (3×CH₃); m/z(EI) 468 (M⁺), 378, 364.

17-Tetrabenzo(a,c,g,i)fluorenylmethyl chloroformate (37)

To a solution of17-(trimethylsilyl)oxymethyl-tetrabenzo(a,c,g,i)fluorene (0.177 g, 0.38mmol) in dichloromethane (5 ml) was added phosgene (1.5 ml, 2.9 mmol;7.5 equiv; 1.93M in toluene). The reaction mixture was heated underreflux for 2 h and then stirred at room temperature for 48 h, undernitrogen. The solvent was removed in vacuo to give compound (37) as ayellow solid which was recrystallized from dichloromethane/n-hexane(36.9 mg, 21%); m.p. 188-189° C.; (Found: C, 80.9; H, 4.29; N, 0.45;C₃₁H₁₉O₂Cl requires: C, 81.1; H, 4.14%); ν_(max) (CH₂Cl₂) 3060 (CHstretching, aromatic), 1775 (CO, chloroformate), 1610, 1500 (aromaticrings), 1160, 1140 (CO stretching) cm⁻¹; δ_(H) (CDCl₃, 200 MHz)8.79-8.60 (6H, m, aromatic), 8.20-8.15 (2H, m, aromatic), 7.76-7.57 (8H,m, aromatic), 5.17 (1H, t, ³J=5.7 Hz, CH), 4.77 (2H, d, ³J=5.7 Hz, CH₂);δ_(c) (CDCl₃, 50 MHz) 150.5 (CO, chloroformate), 140.3, 137.2, 131.6,130.5, 128.2, 127.7 (quaternary aromatic C's), 127.5, 127.0, 126.3,126.2, 125.1, 124.5, 123.5, 123.4 (aromatic CH's), 74.6 (CH₂), 46.5(CH).

EXAMPLE 5 Preparation ofNα-17-tetrabenzo(a,c,g,i)fluorenylmethoxycarbonylglycine (Tbfmoc Gly OH)

Tbfmoc Gly OH was synthesized in three steps from alcohol (31) via thep-nitrophenyl carbonate (41).

(a) para-Nitrophenyl chloroformate (2 equiv), N,N′-dimethylaniline (1equiv), CH₂Cl₂, r.t., 72 h, 80%; (b) CH₃COOH.NH₂CH₂COOC(Me)₃,N,N′-dimethylaniline (2 equiv), CH₂Cl₂, r.t., 24 h, 79%; (c)para-toluenesulfonic acid (0.3 equiv), CH₂Cl₂, reflux, 5 h, 90%.

Reaction of commercially available para-nitrophenyl chloroformate (2equiv) with alcohol (31) and N,N′-dimethylaniline gave the mixedcarbonate (41) in 80% yield. This material was in turn reacted with theacetate salt of glycine tert-butyl ester in the presence of twoequivalents of N,N′-dimethylaniline to afford the protected glycinetert-butyl ester (42) (79%). Simple hydrolysis of the latter usingp-toluenesulphonic acid in refluxing DCM gave the desired acid (36) in90% yield. This is an easy synthesis of Tbfmoc Gly OH using mildly basicconditions in a 57% overall yield based on alcohol (31).

17-Tetrabenzo(a,c,g,i)fluorenylmethyl-p-nitrophenyl carbonate (41)

To a solution of 17-hydroxymethyltetrabenzo-(a,c,g,i)fluorene (0.05 g,0.126 mmol) and p-nitrophenyl chloroformate (35.5 mg, 0.175 mmol; 1.4equiv.) in dichloromethane (2 ml) was added N,N′-dimethylaniline (16 μl,0.126 mmol). The reaction was stirred at room temperature, undernitrogen, for 24 h. The reaction was driven to completion by adding morep-nitrophenyl chloroformate (45 mg, 0.223 mmol; 1.7 equiv.) and bystirring at room temperature for a further 48 h. After purification byflash chromatography using toluene as the eluent, the fractionscontaining material of R_(f)=0.29 were evaporated to give a yellow oil.Trituration in petrol (b.p. 40-60° C.) gave compound (41) as a yellowsolid (56.5 mg, 80%); m.p. 139-140° C.; (Found: C, 77.7; H, 3.94; N,2.29; C₃₇H₂₃NO₅ requires: C, 79.1; H, 4.10; N, 2.49%); t.l.c. R_(f) (C)0.29, R_(f) (A) 0.16; ν_(max) (CH₂Cl₂) 3060 (CH stretching, aromatic),2960, 2930, 2880 (CH stretching, alkyl), 1770 (CO, carbonate), 1620,1600, 1495 (aromatic rings), 1530, 1350 (conjugated nitro-NO₂), 1215 (COstretching), 860 (CH bending, p-disubstituted) cm³¹ ¹; λ_(max) 366(20035) 301 (52092), 289 (48085), 260 (85751), 252 (88957) nm; δH(CDCl₃, 200 MHz) 8.79-8.76 (4H, m, aromatic), 8.61-8.57 (2H, m,aromatic), 8.33-8.16 (2H, m, aromatic), 7.91 (2H, d, J_(AB)=8.9 Hz,p-nitrophenyl), 7.76-7.56 (8H, m, aromatic), 6.64 (2H, d, J_(AB)=8.9 Hz,p-nitrophenyl), 5.14 (1H, t, ³J=4.7 Hz, CH), 4.96 (2H, d, ³J=4.7 Hz,CH); δc (CDCl₃, 50 MHz) 154.9 (CO, carbonate), 151.6 (quaternaryaromatic C_(1′)), 144.9 (quaternary aromatic C_(4′)), 140.3, 137.4,131.5, 130.4, 128.3, 127.7 (quaternary aromatic c's), 127.4, 127.0,126.2, 125.1, 124.7, 124.1, 123.5, 121.2 (aromatic CH's), 125.3(aromatic CH_(2′,6′)), 121.4 (aromatic CH_(3′,5′)), 71.2 (CH₂), 46.8(CH); m/z (EI) 378, 139, 44; (FAB) 561 (M⁺), 379. HRMS 561.15759,C₃₇H₂₃NO₅ requires: 561.15761 ( ) <1 ppm.

Nα-17-Tetrabenzo(a,c,g,i)fluorenylmethoxycarbonyl glycine tert-butylester (42)

To a solution of 17-tetrabenzo(a,c,g,i)fluorenylmethyl-p-nitrophenylcarbonate (39.2 mg, 0.07 mmol) and glycine tert-butyl ester acetate salt(14.7 mg, 0.077 mmol; 1.1 equiv.) in dichloromethane (1.5 ml) was addedN,N′-dimethyl aniline (18 μl, 0.14 mmol; 2 equiv). The reaction mixturewas stirred at room temperature, under nitrogen, for 72 h. Afteraddition of water (10 ml) and acidification with KHSO₄ (2M; pH=1), thereaction mixture was extracted with dichloromethane (3×15 ml), washedwith water (3×20 ml) and dried over MgSO₄. The solvent was removed invacuo to give an orange oil which was triturated in ether. A yellowsolid was obtained which was filtered, washed with petrol (b.p. 40-60°C.) and dried to give compound (42) (30.6 mg, 79%); m.p. 170-171° C.;(Found: C, 79.7; H, 5.51; N, 2.33; C₃₇H₃₁NO₄ requires: C, 80.3; H, 5.60;N, 2.53%); t.l.c. R_(f) (H) 0.79, R_(f) (I) 0.95; ν_(max) (CH₂Cl₂) 3440(secondary amide NH), 3060 (CH stretching, aromatic), 2940 (CHstretching, alkyl), 175 (CO, urethane, ester and slide I), 1600, 1500(aromatic rings), 1520 (amide II), 1340 (OH bonding) cm⁻¹, 1220, 1160,1110 (CO stretching), 865, 850 (out of plane CH bonding); λ_(max) 384(16323), 367 (17656), 302 (42641), 290 (34313), 262 (62296) nm; δH(CDCl₃, 80 MHz) 8.85-8.60 (6H, m, aromatic), 8.39-8.28 (2H, m,aromatic), 7.81-7.56 (8H, m, aromatic), 5.27 (1H, t, ³J=6 Hz, CH), 4.98(1H, s broad, NH), 4.61 (2H, d, ³J=6 Hz, CH₂), 3.74 (2H, d, ³J=6 Hz, CH₂glycine), 1.44 (9H, s, CH₃×3); δc (CDCl₃, 50 MHz) 168.7 (CO, ester),156.1 (CO, urethane), 141.9, 136.7, 131.5, 130.3, 128.7, 127.9(quaternary aromatic C's), 127.4, 126.8, 126.0, 125.8, 125.3, 124.9,123.5, 123.1 (aromatic CH's), 82.0 (quaternary C, CMe₃), 69.0 (CH₂),47.5 (CH), 43.2 (CH₂, glycine), 27.9 (CH₃×3); m/z (FAB) 553, 379. HRMS553.22527, C₃₇H₃₁NO₄ requires 553.22529 ( ) <1 ppm.

Nα-17-Tetrabenzo(a,c,g,i)fluorenylmethoxycarbonyl glycine (Tbfmoc GlyOH) (36)

A solution of Nα-17-tetrabenzo(a,c,g,i)fluorenyl-methoxycarbonyl glycinetert-butyl ester (0.39 g, 0.708 mmol) and p-toluenesulphonic acid (39.2mg, 0.206 mmol) in dichloromethane (15 ml) was heated under reflux for4.5 h, during which time a precipitate was formed. This precipitate wasfiltered, washed with dichloromethane (2×15 ml) and dried to affordcompound (36) (319 mg, 90%); m.p. 240-241° C.; (Found: C, 77.7; H, 4.5;N, 2.0; C₃₃H₂₃NO₄ requires: C, 79.7; H, 4.63; N, 2.82%); t.l.c. Rf (H)0.56, R_(f) (I) 0.80; ν_(max) (KBr disc) 3320 (NH stretching), 3060 (CHstretching, aromatic), 1735 (CO, acid), 1710 (CO, urethane), 1680 (amideI), 1540 (amide II), 1610, 1500 (aromatic rings), 1435 (CH deformations,alkyl), 1310 (OH bending), 1260, 1240,1175, 1160, 1040 (CO stretching),1000, 745, 720 (out of plane CH bending) cm⁻¹; λ_(max) 382 (21744), 368(23297), 303 (55913), 291 (45041); 264 (82005), 255 (88839) nm; δH(d-dioxan, 200 MHz), 9.05-8.97 (4H, 2×d, aromatic), 8.83 (2H, d, ³J=7.8Hz, aromatic), 8.64 (2H, d, ³J=7.8 Hz, aromatic), 7.95-7.74 (8H, m,aromatic), 6.53 (1H, t, ³J=5.8 Hz, NH), 5.61 (1H, t, ³J=5.1 Hz, CH),4.78 (2H, d, ³J=5.1 Hz, CH₂), 3.96 (H, d, ³J=5.8 Hz, CH₂ glycine); δc(d-dioxan, 50 MHz) 170.6 (CO, acid), 156.2 (CO, urethane), 142.5, 136.2,131.3, 130.0, 128.7, 127.7 (quaternary aromatic C's), 127.0, 126.5,125.6, 125.4, 124.5, 123.3, 122.8 (aromatic CH's), 68.3 (CH₂), 47.6(CH), 41.5 (CH₂, glycine); m/z (FAB) 497, 397. HRMS 498.17053, C₃₃H₂₄NO₄requires 498.17052 ( ) <1 ppm. HPLC: column (RP 18), solvents: A(H₂O/TFA(0.1%)), B (CH₃CN/TFA(0.1%)); conditions: B (75%), A (25%);λ=229 nm, AUFS=2 or λ=368 nm, AUFS=1; flow rate: 1 ml/min; injection: 25μl, C=1.3 mg/ml (dioxan/water (1:1)), retention time: 5.4 min.

EXAMPLE 6 Solid Phase Synthesis

All protected amino acid derivatives were purchased from Novabiochem andhave L-stereochemistry. Solid phase peptide synthesis was carried out onan Applied Biosystems 430A peptide synthesizer. The DMF used wassupplied by Rathburn Chemicals Ltd. (peptide synthesis grade). The firstresidue of each sequence (i.e., the C-terminal residue) was coupled tothe p-alkoxybenzyl alcohol resin outside the synthesizer. The extent ofcoupling was determined by deprotecting a small sample of the loadedresin and quantitatively checking the olefin produced by UV at 300 nm inthe case of Fmoc-peptides and at 364 nm in the case of Tbfmoc-peptides.Each residue was coupled twice (2 equiv.), firstly by the symmetricalanhydride method and secondly by the HOBt active ester method (‘doublecoupling’) (except Arg, Asn, Gln double coupled via HOBt ester and Glycoupled once via symmetrical anhydride (4 equiv.). Preformed symmetricalanhydrides were prepared from Fmoc-amino acids (1 mmol) and DICI (0.5mmol) with an activation time of 15 minutes, and HOBt esters fromFmoc-amino acids (0.5 mmol), DICI (0.5 mmol) and HOBt (0.5 mmol) with anactivation time of 30 minutes. Coupling reactions were carried out overa period of 30-60 minutes. Capping of unreacted amino functions wasperformed for 6 minutes using acetic anhydride and pyridine in DMF.Deprotection of the Fmoc-peptide-resin was achieved over a 12 minuteperiod (5+3+3+1 minutes) using a solution of 20% piperidine in DMF. Theresin was washed thoroughly with DMF at the end of each cycle. As arough monitor of the couplings, the product solution from thedeprotection steps was fed through an ultraviolet detector (313 nm) andits absorption recorded to give a series of peaks. Removal of peptidesfrom the resin and simultaneous cleavage of side chain protecting groupswere performed using a mixture of trifluoroacetic acid/water/scavenger(95:5:5) for 2-3 hours. Chromatography of Nα-protected peptides wascarried out using graphitized carbon (PGC 220-224; 150-180 pm, 100 m²/g)in a glass column. HPLC was carried out on Waters system using an ABIaquapore Prep 10 C-18 300 Å pore size 20 μm spherical silica (10 mmID×250 mm) column for preparative separations and an ABI RP 18 aquaporeOD 300 7 μm spherical silica (4.6 mm ID×220 mm) column for analyticalseparations. A gradient was used, as specified in parentheses, betweensolvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile).The flow rate was 1 ml/min for analytical HPLC and 5 ml/min forpreparative HPLC. Elution of the samples was monitored by ultravioletabsorption at 14 nm. Amino acid analyses were carried out on a LKB 4150alpha amino acid analyzer following sealed tube hydrolysis in constantboiling hydrochloric acid at 110° C. for 18-36 hours.

Synthesis of Fmoc Gly Ser Met Val Leu Ser OH SEQ ID NO:1 and Tbfmoc GlySer Met Val Leu Ser OH SEQ ID NO:1 and Comparison of Properties Thereofa. Synthesis of Fmoc Gly Ser Met Val Leu Ser OH SEQ ID NO:1

The protected resin-bound peptide Fmoc Ser (O^(t)Bu) Met Val Leu Ser(O^(t)Bu) SEQ ID NO:2 (43) was prepared on a peptide synthesizer usingan orthogonal strategy. Fmoc was used for the temporary protection ofthe amine function of each amino acid and was removed before eachcoupling with a 20% piperidine in DMF solution. The side-chain functionof serine was protected with the t-butyl group. The coupling proceduresinvolved a symmetrical anhydride followed by an HOBt ester coupling.Acetic anhydride and pyridine were used to acetylate any unreacted aminofunctions. The Merrifield resin was employed with thep-alkoxybenzylalcohol group as the linker.

Fmoc Gly Ser Met Vat Leu Ser OH SEQ ID NO:1 (44) was synthesized fromthe resin-bound peptide (43) via the series of reactions outlined below.

After deprotection of the Fmoc group under basic conditions theamino-terminus of the peptide SEQ ID NO:2 was coupled in dioxan to FmocGly OH via its symmetrical anhydride (3 equiv) with a couplingefficiency of 88%. Cleavage of the hexapeptide SEQ ID NO:1 from theresin and removal of the t-butyl groups was carried out using TFA, andin the presence of ethylmethylsulphide and anisole as cation scavengers.After removal of the solvent in vacuo the peptide SEQ ID NO:1 wasfinally precipitated with ether, filtered off and dried. Purificationusing HPLC was carried out using dioxan in which the peptide SEQ ID NO:1was soluble at approximately 1.5 mg/ml. Preparative HPLC gave thepeptide SEQ ID NO:1 (44) together with a trace of the sulfoxidederivative, as indicated by mass spectrometry (MH+16⁺). The identity ofthe peptide SEQ ID NO:1 was confirmed by mass spectrometry and aminoacid analysis.

Nα-9-Fluorenylmethoxycarbonyl glycylserylmethionylvalylleucylserine,Fmoc Gly Ser Met Val Leu Ser OH SEQ ID NO:1 (44)

The resin-bound peptide Fmoc Ser (O^(t)Bu) SEQ ID NO:2 Met Val Leu Ser(O^(t)Bu) (130.5 mg, 0.05 mmol) was sonicated in a solution of 20%piperidine in N,N-dimethylformamide (5 ml) for 20 min., then filteredand finally washed well with N,N-dimethyl-formamide, dichloromethane anddioxan. Fmoc Gly OH (89.2 mg, 0.3 mmol; 6 equiv.) was dissolved indioxan (2 ml); 1,3-diisopropylcarbodiimide (47 μl, 0.3 mmol; 6 equiv.)was then added and the reaction mixture was sonicated for 5 min. Thissolution was then added to the resin-bound peptide SEQ ID NO:2previously swollen in dioxan (1 ml). The reaction mixture was sonicatedfor 4.5 h. The resin-bound peptide SEQ ID NO:1 was filtered, washed withdichloromethane, ether, and dried (129.8 mg); product resinfunctionality in mmol/g: 0.334, resin loading/coupling percentage: 88;A₃₀₀ nm=0.76 (resin-peptide SEQ ID NO:1: 2.52 mg). To a mixture ofresin-peptide (126.1 mg, 0.042 mmol) anisole (0.25 ml),ethylmethylsulphide (0.5 ml) and water (0.25 ml) was addedtrifluoroacetic acid (10 ml). The reaction mixture was sonicated for 2h. The resin was filtered, washed with trifluoroacetic acid (2×1 ml) andchloroform (4 ml). The solvent was removed in vacuo to give a residue.The peptide SEQ ID NO:1 was precipitated on addition of ether, filtered,washed with ether and dried (42.7 mg); (Found: Ser₂ 1.90, Gly₁ 1.05,Val₁ 0.99, Met₁ 1.02, Leu₁ 1.01); m/z (FAB) 853, 837, 815 (MH⁺). HRMS815.36488, C₃₉H₅₅N₆O₁₁S₁ requires: 815.36492 ( ) <1 ppm. HPLC: column RP18, solvents: A (H-₂O/TFA (0.1%)), B (CH₃CN/TFA (0.1%)); conditions:

λ=229 nm; AUFS=0.2; flow rate: 1 ml/min; injection: 25 μl (C=0.4 mg/0.5ml (dioxan)); retention time: 14.2 min.

b. Synthesis of Tbfmoc Gly Ser Met Val Leu Ser OH SEQ ID NO:1

Tbfmoc Gly Ser Met Val Leu Ser OH SEQ ID NO:1 (45) was then preparedfrom the resin-bound peptide SEQ ID NO:2 (43) via a similar method.

Tbfoc Gly OH was coupled in dioxan with an 80% coupling efficiency.Removal of the peptide SEQ ID NO:1 from the resin and t-butyl cleavagewere carried out with TFA in the presence of the same scavengers. Thepeptide SEQ ID NO:1 was finally precipitated with ether, filtered offand dried (69% yield).

The peptide SEQ ID NO:1 was purified as before. A fresh solution of thecrude peptide SEQ ID NO:1 in dioxan gave one peak on HPLC, but onstanding (30 min.) the same solution gave two peaks corresponding to thedesired peptide SEQ ID NO:1 and the sulfoxide derivative. The facileoxidation of methionine to the sulfoxide might be due to the presence ofperoxides in the dioxan. After isolation of these two peptides SEQ IDNO:1 by preparative HPLC, these assignments were confirmed by massspectrometry (MH⁺ and MH+16⁺) and amino acid analysis.

Nα-17-Tetrabenzo(a,c,g,i)fluorenylmethoxycarbonylglycylserylmethionylvalylleucylserine(Tbfmoc Gly Ser Met Val Leu Ser OH) SEQ ID NO:1 (45)

The resin-bound peptide Fmoc Ser (O^(t)Bu) Met Val Leu Ser (O^(t)BU) SEQID NO:2 (65.3 mg, 0.025 mmol) was sonicated in a solution of 20%piperidine in DMF (5 ml) for 20 min. The resin-bound peptide SEQ ID NO:2was then filtered and washed well with DMF, dichloromethane and dioxan.Tbfmoc Gly OH (74.5 mg, 0.15 mmol; 6 equiv.) was sonicated for 30 min.in dioxan (2 ml). 1,3-Diisopropylcarbodiimide (23.5 μl, 0.15 mmol; 6equiv.) was then added and the reaction mixture was sonicated for 5 min.This solution was then added to the resin-bound peptide SEQ ID NO:2previously swollen in dioxan (1 ml). The reaction mixture was sonicatedfor 5 h. The resin was filtered, washed well with dichloromethane,dioxan and ether, and then dried (58.3 mg); product resin functionalityin mmol/g: 0.28; resin loading/coupling percentage: 80; A₃₆₄ nm=0.43(resin-bound peptide SEQ ID NO:1: 0.85 mg). To a mixture of resin-boundpeptide (57.3 mg, 0.016 mmol), anisole (0.5 ml), ethylmethylsulphide(0.25 ml) and water (0.5 ml), was added trifluoroacetic acid (5 ml). Thereaction mixture was sonicated for 2 h. The resin was filtered andwashed with trifluoroacetic acid (1 ml) and chloroform (4 ml) and thesolvent was removed in vacuo. Trituration of the residue in ether gavethe desired peptide SEQ ID NO:1 as a yellow solid which was filtered,washed well with ether and dried (17.2 mg, 69%); (Found: Ser₂ 1.8, Gly₁0.08, Val₁ 1.00, Met₁ 0.96, Leu₁ 1.00; m/z (FAB) 1053, 1037, 1015 (MH⁺).HRMS 1015.42761, C₅₅H₆₃N₆O₁₁S₁ requires: 1015.42752 ( ) <1 ppm. HPLC:column RP18, solvents: A (H₂O/TFA (0.1%)); B (CH₃CN/TFA (0.1%);conditions: A;

λ=229 nm; AUFS=1; flow rate: 1 ml/min; injection: 25 μl (C=0.2 mg/0.2 ml(dioxan)); retention time: 16.1 min.

c. Behavior of Tbfmoc and Fmoc Derivatives on PGC

The retention of both Fmoc Gly OH and Tbfmoc Gly OH was compared on acolumn packed with graphitized carbon.

A solution of Fmoc Gly OH (10.2 mg, 34.3 μmole) in dioxan was loaded ona 6 mm diameter column packed with PGC (1.5 g). Only 15 ml of dioxan wasrequired to elute the compound completely (as monitored by t.l.c.). Incontrast, when Tbfmoc Gly OH (10.2 mg, 20.5 μmole) was loaded under thesame conditions, none of this material was eluted in the first 35 ml.Indeed the total volume of dioxan needed to elute the compound was 300ml.

The behavior on a graphitized carbon column of Fmoc Gly Ser Met Val LeuSer OH SEQ ID NO:1 (44) was then compared with the Tbfmoc derivative SEQID NO:1 (45). To increase the overall retention time, a mixture ofdioxan/water (2:1) was used to elute both peptides. After loading asolution of the Fmoc hexapeptide SEQ ID NO:1 (9.2 mg, 11.3 μmole) in amixture of dioxan/water (2:1) onto the column, the peptide SEQ ID NO:1was eluted completely with 75 ml of the solvent mixture. In contrast,the Tbfmoc derivative SEQ ID NO:1 (10.5 mg, 10.3 μmole) was not elutedat all in the first 80 ml of solvent and was subsequently only veryslowly eluted from the column.

d. Deprotection of the Tbfmoc Group

Deprotection of the Tbfmoc group from the peptide, while still retainedon the graphitized carbon column was carried out.

The deprotection was carried out using 20% piperidine in a mixture ofdioxan/water (2:1). Under these conditions the Tbfmoc hexapeptide SEQ IDNO:1 was totally deprotected. The free peptide (H Gly Ser Met Val LeuSer OH SEQ ID NO:1 (46)) was eluted and was monitored by t.l.c. usingninhydrin to reveal the amino function. The subsequent isolation of theproduct was simple; the piperidine and solvents were removed in vacuoand the peptide SEQ ID NO:1 was precipitated with ether, filtered anddried (85%). Mass spectrometry showed both the presence of the peptideSEQ ID NO:1 (MH⁺) and the sulfoxide derivative (MH+16⁺). Amino acidanalysis confirmed the composition of the peptide SEQ ID NO:1.

Glycylserylmethionylvalylleucylserine (H Gly Ser Met Val Leu Ser OH) SEQID NO:1 (46)

A solution of Tbfmoc Gly Ser Met Val Leu Ser OH SEQ ID NO:1 (29.2 mg,8.8 μmol) in a mixture of dioxan and water (2:1; 15 ml) was loaded on a9 mm diameter column packed with graphitized carbon (4 g). The columnwas then eluted with a mixture of dioxan and water (2:1; 60 ml). Thedeprotection was carried out by eluting the column with 20% piperidinein a mixture of dioxan and water (2:1; 20 ml). The fractions containingthe peptide SEQ ID NO:1 (monitored by t.l.c., R_(f)=0(MeOH/CHCl₃/CH₃COOH (1:9:0.5)) using ninhydrin) were combined andevaporated to give a residue. The peptide SEQ ID NO:1 was precipitatedwith ether, filtered and dried (14.5 mg, 85%); (Found: Ser₂ 1.92, Gly₁0.98, Val₁ 1.03, Met₁ 0.87, Leu₁ 1.02; m/z (FAB) 609, 593 (MH⁺). HRMS593.29689, C₂₄H₄₅O₉N₆S requires: 593.29685 ( ) <1 ppm.

Finally, the tetrabenzofluorene olefin or its piperidine adduct, theby-products from the deprotection step, were removed from the column byeluting with hot dioxan.

EXAMPLE 7 Synthesis of Fmoc Ubiquitin (54-76) OH SEQ ID NO:4 and TbfmocUbiquitin (53-76) OH SEQ ID NO:3 and Comparison of Properties Thereof a.Synthesis of Fmoc Ubiquitin (54-76) OH SEQ ID NO:4

The resin-bound peptide Fmoc ubiquitin (54-76) SEQ ID NO:4 (47) wasprepared on a peptide synthesizer under the conditions described inExample 6(a). Most coupling procedures involved a symmetrical anhydridefollowed by an HOBt ester coupling in a mixture of DMF/dioxan (1:1) asthe solvent.

b. Synthesis of Tbfmoc Ubiquitin (53-76) OH SEQ ID NO:3

Tbfmoc ubiquitin (53-76) OH SEQ ID NO:3 (48) was synthesized from theresin-bound peptide SEQ ID NO:4 (47) as depicted below.

The resin-bound peptide Fmoc ubiquitin (54-76) SEQ ID NO:4 was initiallytreated with a mixture of acetic anhydride/pyridine (1:1) in order tocap any amino functions. After deprotection in basic conditions thepeptide SEQ ID NO:4 was coupled in dioxan to Tbfmoc Gly OBt ester (4equiv), generated in situ from DIC and HOBt, with a coupling efficiencyof 88%. After final cleavage using TFA in the presence of anisole andthioanisole as cation scavengers, the Tbfmoc ubiquitin peptide SEQ IDNO:3 (48) was precipitated with ether (96% yield).

Nα-(17-Tetrabenzo(a,c,g,i)fluorenylmethoxycarbonyl)glycylarginylthreonylleucylseryllaspartyltyrosylasparagylisoleucylglutaminyllysylglutamylserylthreonylleucylhistidylleucylvalylleucylarginylleucylarginylglycylglycine (Tbfmoc-ubiquitin(53-76) OH) SEQ ID NO:3 (48)

To the Fmoc-ubiquitin (54-76) SEQ ID NO:4 resin bound peptide (150 mg,2.65 μmol) in DMF (1 ml) was added acetic anhydride (21.4 μl, 226 μmol;10 equiv.) and pyridine (18.2 μl, 226 μmol; 10 equiv.). The reactionmixture was sonicated for 30 min. After filtration and washing with DMFand ether, the resin-bound peptide SEQ ID NO:4 was sonicated for 15 min.in a solution of 20% piperidine in DMF (25 ml). The resin bound peptideSEQ ID NO:4 was then filtered and washed well with DMF and ether. TbfmocGly OH (45 mg, 0.09 mmol; 4 equiv.) was sonicated for 30 min. in dioxan(1.5 ml) until complete dissolution. To this solution was added1,3-diisopropylcarbodiimide (14 μl, 0.09 mmol; 4 equiv.) and1-hydroxybenzotriazole (12.2 mg, 0.09 mmol; 4 equiv.). The reactionmixture was sonicated for 2 min. This solution was then added to theresin bound peptide SEQ ID NO:4 previously swollen for 30 min. in dioxan(1.5 ml). After sonication for 3.5 h, the resin bound peptide SEQ IDNO:3 was filtered, washed with dioxan and ether and dried overnight in adesiccator under vacuum (148.1 mg).

Substitution Efficiency

The Tbfmoc resin bound peptide SEQ ID NO:3 (2.1 mg) was sonicated for 30min. in a solution of 20% triethylalmine in DMF (10 ml). The specificabsorption at 364 nm was recorded and the following results were derivedfrom it: product resin functionality in mmol/g: 0.133; resinloading/coupling percentage: 88; A₃₆₄ nm=0.505 (resin bound peptide: 2.1mg). To the resin bound Tbfmoc-peptide SEQ ID NO:3 (146 mg) was addedwater (75 μl), thioanisole (75 μl), anisole (75 μl) and trifluoroaceticacid (3 ml). The reaction mixture was sonicated for 2.5 h. The resin wasfiltered, washed with TFA (2×0.5 ml) and chloroform (2×1 ml), and dried(24 mg). The filtrate was evaporated under reduced pressure to give aresidue. Trituration in ether gave the crude peptide SEQ ID NO:3 as ayellow solid which was chilled overnight, filtered and dried (68.5 mg).A suspension of crude Tbfmoc-ubiquitin SEQ ID NO:3 (53-76) (12 mg) inCH₃CN/H₂O (1:1; 0.1% TFA; 12 ml) was then sonicated for 2 h untilcomplete dissolution had occurred. Purification of this product wasfinally achieved by preparative HPLC using a reverse phase C-18 column(10×250 mm) with a gradient of A:B, 20 to 80% B over 25 min. andultraviolet detection at 214 nm, to yield pure Tbfmoc ubiquitin SEQ IDNO:3 (53-76) after lyophilization as a white powder (4.5 mg); amino acidanalysis: Asx₂ 2.14, Thr₂ 1.94, Ser₂ 1.87, Glx₂ 2.38, Gly₃ 2.73, Val₁1.14, Ile₁ 0.94, Leu₅ 5.02, Tyr₁ 0.94, His₁ 1.01, Lys₁ 0.94, Arg₃ 2.94;m/z (FAB) 3147.4 (MH⁺), HRMS 3149.64063, C₁₄₉H₂₁₉N₃₈O₃₈ requires:3149.64048 ( ) <1 ppm;

214 nm; C=0.2 mg/0.2 ml (A), injection: 25 μl) R_(t)=18.2 min.

c. Behavior of Nα-Acetyl-Ubiquitin (54-76) OH SEQ ID NO:4 and TbfmocUbiquitin (53-76) OH SEQ ID NO:3 on PGC

Nα-Acetyl-ubiquitin (54-76) OH SEQ ID NO:4 (49) was prepared from theresin-bound peptide SEQ ID NO:4 (47) by the following reactions.

Deprotection of the Fmoc group from the peptide SEQ ID NO:4 using 20%piperidine in DMF gave the peptide SEQ ID NO:4 with a free N-terminalamino group, which was then acetylated with a mixture of aceticanhydride/pyridine (1:1) (100 equiv). Removal of the peptide SEQ ID NO:4from the resin and cleavage of the side-chain protecting groups werecarried out using TFA in the presence of anisole and thioanisole asscavengers. Subsequent precipitation with ether afforded crudeNα-acetyl-ubiquitin (54-76) OH SEQ ID NO:4 (49).

Nα-(Acetyl)arginylthreonylleucylserylaspartyltyrosylasparagylisoleucylglutaminyllysylglutamylserylthreonylhistidylleucylvalylleucylarginylleucylarginylglycylglycine,Nα-acetyl-ubiquitin (54-76) OH SEQ ID NO:4 (49)

The resin bound peptide Fmoc-ubiquitin SEQ ID NO:4 (54-76) (100 mg,0.0151 mol) was sonicated for 30 min. in a solution of 20% piperidine inDMF (25 ml) then filtered, and finally washed well with DMF and ether.To the resin bound peptide SEQ ID NO:4 previously swollen for 30 min. inDMF (1 ml) was added acetic anhydride (143 μl, 1.51 mmol; 100 equiv.)and pyridine (122 μl, 1.51 mmol; 100 equiv.). The reaction mixture wassonicated for 1 h. The resin-bound peptide SEQ ID NO:4 was filtered andwashed with DMF and ether. To the resin bound Nα-acetyl-peptide SEQ IDNO:4 was added water (50 μl), thioanisole (50 μl), anisole (50 μl) andtrifluoroacetic acid (2 ml). The reaction mixture was sonicated for 2.5h. The resin was filtered, washed with TFA (2×0.5 ml) and chloroform(2×0.5 ml). The solvent was removed in vacuo. Trituration in ether gavethe product as a white precipitate which was chilled overnight, filteredand dried (43.6 mg). Purification of this peptide SEQ ID NO:4 (23.4 mg)was by preparative HPLC using the same reverse phase C-18 column with agradient of A:B, 10 to 60% B over 16 min. and UV detection at 214 nm, togive pure Nα-acetyl-ubiquitin (54-76) OH SEQ ID NO:4 as a white solidafter lyophilization (9.7 mg); amino acid analysis: Asx₂ 1.98, Thr₂1.82, Ser₂ 1.69, Glx₂ 2.33, Gly₂ 2.36, Val₁ 1.14, Ile₁ 0.91, Leu₅ 5.06,Tyr₁ 0.85, His₁ 1.01, Lys₁ 0.98, Arg₃ 2.87; m/z (FAB) 2712.1 (MH⁺), HRMS2712.49903, C₁₁₈H₂₀₀N₃₇O₃₆ requires 2712.49891 ( ) <1 ppm;

214 nm; C=0.2 mg/0.2 ml (A), injection: 25 μl), R_(t)=13.2 min.

The behavior of both the Nα-acetyl SEQ ID NO:4 and Tbfmoc ubiquitin SEQID NO:3 peptides on a PGC column was then examined.

A solution containing 5 mg of each peptide was loaded onto the columnwhich was then eluted using different solvent mixtures. After collectingthe fractions, the solvent was removed in vacuo and the residue obtainedwas redissolved in fresh solvent before analysis by HPLC.

The best results were obtained when a mixture of CH₃CN/H₂O (1:1) wasused. Under these conditions, the Nα-acetyl-peptide SEQ ID NO:4 waseluted completely by 30 ml of the mixture, whereas the Tbfmoc peptideSEQ ID NO:3 was retained.

The total chromatographic separation of these closely related peptidesclearly demonstrated the efficiency of the method and its potential forthe purification of peptides synthesized by solid phase methodology,provided careful attention is paid to the choice of solvents.

d. Purification of Ubiquitin (53-76) OH SEQ ID NO:3

A simple chromatographic elution on a column packed with graphitizedcarbon was used as the first purification step (as described in (c)above). Crude Tbfmoc ubiquitin (53-76) OH SEQ ID NO:3 (48) dissolved ina mixture of CH₃CN/H₂O (1:1; 0.5% TFA) was loaded onto a column packedwith PGC (50×mass of peptide). 50 ml of a mixture of the same solventwas required to completely elute the main impurities (probably Nα-acetylubiquitin SEQ ID NO:7 (55-76) and unreacted ubiquitin SEQ ID NO:4(54-76)). None of the Tbfmoc peptide SEQ ID NO:4 was eluted even afterflushing the column with a further 50 ml of CH₃CN/H₂O (1:1).Deprotection of the Tbfmoc group was carried out using 20% piperidine ina mixture of CH₃CN/H₂O (1:1) and only the desired peptide, ubiquitin(53-76) OH SEQ ID NO:3, was eluted. After removal of the solvent invacuo, the peptide SEQ ID NO:3 was finally precipitated with ether.Subsequent purification by preparative HPLC gave pure ubiquitin (53-76)OH SEQ ID NO:3 (50) in 15% overall yield.

Glycylarginylthreonylleucylserylaspartyltyrosylasparagylisoleucylglutaminyllysylglutamylserylthreonylleucylhistidylleucylvalylleucylarginylleucylarginylglycylglycine (ubiquitin (53-76)OH) SEQ ID NO:3

A solution of Tbfmoc-ubiquitin (53-76) OH SEQ ID NO:3 (30 mg; crudepeptide) in a mixture of CH₃CN/H₂O (1:1; 0.5% TFA; 30 ml) was sonicatedfor 30 min. until complete dissolution and loaded on a 5 mm diameterglass column packed with graphitized carbon (1.4 g; PGC 220-224; 150-180μm; 100 m²/g; length after packing: 140 mm). The column was first elutedwith a mixture of CH₃CN/H₂O (1:1; 0.5% TFA; 50 ml). After lyophilizationa residue (6.1 mg) was obtained which gave a major peak on HPLC(R_(t)=13 min., Nα-acetyl-ubiquitin SEQ ID NO:7 (55-76) or ubiquitin SEQID NO:4 (54-76)). The column was then eluted with a mixture of CH₃CN/H₂O(1:1; 50 ml). A residue (0.4 mg) was obtained after lyophilization whichwas found HPLC to contain impurities. The deprotection was carried outusing a 20% piperidine solution in a mixture of CH₃CN/H₂O (1:1; 50 ml).The solvent was removed in vacuo to give a residue which was trituratedin ether, filtered and dried (11 mg). This crude peptide SEQ ID NO:3 (11mg) was finally purified by preparative HPLC (reverse phase C-18 column(10×250 mm); gradient (A:B), 10 to 60% B over 25 min.; detection at 214nm) to give ubiquitin (53-76) OH SEQ ID NO:3 as a white solid afterlyophilization (4.5 mg, 15%); amino acid analysis: Asx₂ 2.04, Thr₂ 1.88,Ser₂ 1.67, Glx₂ 2.27, Gly₃ 3.37, Val₁ 1.01, Ile₁ 0.90, Leu₅ 4.99, Tyr₁0.93, His₁ 1.01, Lys₁ 0.96, Arg₃ 2.95; m/z (FAB) 2727.8 (MH⁺), HRMS2727.50986, C₁₁₈H₂₀₁N₃₈O₃₆ requires: 2727.50981 ( ) <1 ppm;

214 nm; C=0.4 mg/0.4 ml (A), injection: 25 μl), R_(t)=12.6 min.

The identities of both Tbfmoc ubiquitin (53-76) OH SEQ ID NO:3 andubiquitin (53-76) OH SEQ ID NO:3 peptides were established by highresolution mass spectrometry and amino acid analysis.

EXAMPLE 8 Synthesis and Purification of Ubiquitin (35-76) SEQ ID NO:5

The purification of this peptide did not pose any problems and wascarried out as with the smaller ubiquitin fragment SEQ ID NO:3 (50);Tbfmoc ubiquitin (35-76) OH SEQ ID NO:5 (52) was prepared by coupling insitu Tbfmoc Gly OBt to the resin-bound peptide ubiquitin (36-76) SEQ IDNO:3 (75% yield). Following chromatography on a PGC column andpreparative HPLC, the desired peptide SEQ ID NO:5 (51) could beobtained.

The authenticity of (51) was also confirmed by mass spectrometry andamino acid analysis, and its purity by analytical HPLC.

Nα-(17-Tetrabenzo(a,c,g,i)fluorenylmethoxycarbonyl)glycylisoleucylprolylprolylaspartylglutaminylglutaminylarginylleucylisoleucylphenylalanylalanylglycyllysylglutaminylleucyglutamylaspartylglycylarginylthreonylleucylserylaspartyltyrosylasparaglylisoleucylglutaminyllysylglutamylserylthreonylleucylhistidylleucylvalylleucylarginylleucylarginylglycylglycine(Tbfmoc ubiquitin (35-76)) SEQ ID NO:5 (52)

To the Fmoc ubiquitin (36-76) resin-bound peptide SEQ ID NO:6 (109.3 mg,7.88 μmol) in DMF (1 ml) was added acetic anhydride (14.8 μl, 157.6μmol; 20 equiv) and pyridine (12.8 μl, 157.6 μmol; 20 equiv). Thereaction mixture was sonicated for 1 h. After filtration and washingwith DMF and ether, the resin bound peptide SEQ ID NO:6 was sonicatedfor 15 min. in a solution of 20% piperidine in DMF (25 ml). The resinbound peptide SEQ ID NO:6 was then filtered and washed well with DMF andether. Tbfmoc GlyOH (19.6 mg, 39.4 μmol; 5 equiv) was sonicated for 30min. in dioxan (1 ml). To this solution was added1,3-diisopropylcarbodiimide (6.2 μl, 39.4 μmol; 5 equiv) and1-hydroxybenzotriazole (5.3 mg, 39.4 μmol; 5 equiv). The reactionmixture was sonicated for 5 min. This solution was then added to theresin-bound peptide SEQ ID NO:6 previously swollen in dioxan (1 ml) for1 h. The reaction mixture was sonicated for 19 h. The resin-boundpeptide SEQ ID NO:5 was filtered, washed with dioxan, dichloromethaneand ether, and finally dried for 48 h in a vacuum desiccator (102 mg).

Substitution Efficiency

The Tbfmoc resin bound peptide SEQ ID NO:5 (2.9 mg) was sonicated for 30min. in a solution of 20% triethylamine in DMF (10 ml). The specificabsorption at 364 nm was recorded and the following results were derivedfrom it: A=0.65; product resin functionality in μmol/g: 50.51; resinloading/coupling percentage: 70. The coupling was then repeated underthe same conditions (4 equivalents of each Tbfmoc GlyOH,1,3-diisopropylcarbodiimide and 1-hydroxybenzotriazole; reaction time:3.5 h). The resin bound peptide SEQ ID NO:5 was filtered, washed withdioxan, dichloromethane and ether, and finally dried overnight in avacuum desiccator (99.5 mg).

Substitution Efficiency

A=0.265 (resin-bound peptide: 2.7 mg); product resin functionality inμmol/g: 54.26; resin loading/coupling percentage: 75.

To the resin-bound Tbfmoc-peptide SEQ ID NO:5 (96.8 mg) was added water(75 μl), thioanisole (50 μl) and TFA (2.1 ml). The reaction mixture wassonicated for 2.5 h. The resin was filtered, washed with TFA (2×0.5 ml)and CHCl₃ (2×1 ml), and dried (19.1 mg). The solvent was removed invacuo to give a residue. Trituration in diethyl ether gave the crudepeptide SEQ ID NO:5 as a yellow solid which was chilled overnight,washed well with ether and finally dried (49.2 mg). HPLC (A:B; A (90%)

A (10%, 214 nm; C=0.5 mg/0.5 ml (A/B (1:1)); injection 30 μl) R_(t)=19.2min.

Glycylisoleucylprolylprolylaspartylglutaminylglutaminylarginylleucylisoleucylphenylalanylalanylglycyllysylglutaminylleucylglutamylaspartylglycylarginylthreonylleucylserylspartyltyrosylasparagylisoleucylglutaminyllysylglutamylserylthreonylleucylhistidylleucyvalylleucylarginylleucylarginylglycylglycine (ubiquitin(35-76) OH) SEQ ID NO:5 (51)

A suspension of crude Tbfmoc-ubiquitin (35-76) OH SEQ ID NO:5 (30 mg) ina mixture of CH₃CN/H₂O (1:1; 0.5% TFA, 25 ml) was sonicated or 20 min.(until complete dissolution) and loaded on a 5 mm diameter glass columnpacked with graphitized carbon (1.5 g; PGC 220-224; 150-180 μm; 100m²/g; active length: 15 cm). The column was first eluted with a mixtureof CH₃CN/H₂O (1:1; 0.5% TFA; 50 ml). After lyophilization of the eluenta residue (2 mg) was obtained which gave a broad peak on HPLC(R_(t)=13.8 min. (impurities)). The column was then eluted with amixture of CH₃CN/H₂O (1:1; 50 ml). A white solid (4.5 mg) was obtainedafter lyophilization which was found by HPLC also to contain impurities(broad peak, R_(t)=14.1 min.). The column was again eluted with 50 ml ofa mixture of CH₂CN/H₂O (1:1) and a residue (0.3 mg) was obtained afterlyophilization (R_(t)=14.2 min.). The deprotection was carried out usinga 20% piperidine solution in a mixture of CH₃CN/H₂O (1:1; 50 ml) and wasfollowed by an elution of the column with CH₃CN/H₂O (1:1; 50 ml). Thesolvent was removed in vacuo to give a brown residue. Diethyl ether wasadded and the precipitate obtained was chilled overnight, filtered andwashed well with ether, and finally dried (11.7 mg). The crude peptideSEQ ID NO:5 (24.8 mg) was finally purified by preparative HPLC (reversephase C-18 column (10×250 nm); gradient (A:B), 0 to 60% B over 22 min.;detection at 214 nm) to give pure ubiquitin (35-76) OH as a white solidafter lyophilization (4.4 mg); amino acid analysis: Asx₄ 3.98, Thr₂1.83, Ser₂ 1.75, Glx₆ 6.67, Pro₂ 1.91, Gly₅ 5.10, Ala₁ 0.96, Val₁ 1.01,Ile₃ 2.99, Leu₇ 7.00, Tyr₁ 0.94, Phe₁ 1.04, His₁ 1.11, Lys₂ 1.91, Arg₄3.81; m/z (FAB) 4734.2 (MH⁺). HRMS 4734.57186, C₂₀₈H₃₄₄N₆₃O₆₃ requires:4734.57161 ( ) <1 ppm;

λ=214 nm; C=0.1 mg/0.1 ml (A);

injection: 12 μl), R_(t)=3.8 min.

EXAMPLE 9 Synthesis of Tbfmoc-L-Phe OH

The free amine function of commercially available L-phenylalaninetert-butyl ester hydrochloride was initially liberated withtriethylamine. The amine in 20% excess was then reacted with the mixedcarbonate (41) in the presence of N,N′-dimethylaniline. After removal ofthe t-butyl group with TFA, compound (53) was obtained.

The formation of (53) may be rationalized by β elimination of the mixedcarbonate (41), 1,2 addition of the amino ester onto the resultingolefin (38) and finally cleavage of the t-butyl ester.

Nα-17-Tetrabenzo(a,c,g,i)fluorenylmethyl-L-phenylalanine (Tbfm-L-Phe OH)(53)

A solution of Nα-17-tetrabenzo(a,c,g,i)fluorenylmethyl-L-phenylalaninetert-butyl ester (421.1 mg, 0.702 mmol) in trifluoroacetic acid (3.6 ml)and water (0.2 ml) was sonicated for 3.5 h at room temperature. Thesolvent was removed in vacuo to give a brown residue. Trituration indiethylether gave the compound (53) as a yellow solid which was chilledovernight, filtered, washed with ether, and finally dried (352.7 mg,92%);

m.p. 178-180° C.; (Found: C, 85.2; H, 5.47; N, 2.57; C₃₉H₂₉NO₂ requires:C, 86.2; H, 5.37; N, 2.59%); [α]D −40.4° (C=1, TFA); t.l.c. R_(f) (H)0.36, R_(f) (T) 0.62; ν_(max) (KBr) 3090, 3060, 3030 (CH stretching,aryl), 1620 (COOΘ), 1500 (aromatic rings), 1240, 1190, 1040 (COstretching), 750, 725, 700 (CH out-of-plane deformation) cm⁻¹; λ^(max)384 (18868), 369 (21267), 304 (52447), 292 (41894), 264 (78991), 255(86346) nm; δC (TFA, 50 MHz) 169.5 (CO, acid), 137.9, 136.9, 136.1,134.2, 131.8, 131.4, 130.5, 129.8, 126.3, 126.1, 125.8 (quaternaryaromatic C's) 128.7, 127.9, 127.2, 127.0, 126.8, 124.8, 123.44, 123.34,123.29, 122.2 (aromatic CH's), 62.2 (α CH), 50.9 (CH₂), 43.2 (CH), 34.2(β CH₂); m/z (FAB) 544 (MH³⁰ ), 379. HRMS 544.22762, C₃₉H₃₀NO₂ requires:544.22764 ( ) <1 ppm.

As the acetate of glycine was successful in the synthesis of Tbfmoc GlyOH, this method is also useful in this case; thus the acetate salt ofL-phenylalanine tert-butyl ester was prepared.

The hydrochloride salt of the amino acid ester was neutralized withtriethylamine and the precipitated Et₃N.HCl generated was filtered off.Following removal of the solvent, the residue obtained was redissolvedin acetic acid before lyophilization. Subsequent reaction with the mixedcarbonate (41) in the presence of two equivalents ofN,N′-dimethylaniline afforded Tbfmoc-L-Phe O^(t)Bu (54) in 46% yieldafter purification by flash chromatography and recrystallization. Finalcleavage of the ^(t)-butyl ester using a mixture of TFA/H₂O (95:5) gaveTbfmoc-L-Phe OH (55) in excellent yield.

Nα-17-Tetrabenzo(a,c,g,i)fluorenylmethoxycarbonyl-L-phenylalaninetert-butyl Ester (Tbfmoc Phe O^(t)-Bu) (54)

To a suspension of L-phenylalanine tert-butyl ester hydrochloride (126.7mg, 0.491 mmol) in ethyl acetate (6 ml) was added triethylamine (68.5μl, 0.491 mmol). The reaction mixture was stirred for 2.5 h at roomtemperature. The precipitated triethylamine hydrochloride was filteredoff, washed with ethyl acetate (2×0.5 ml) and dried (65.3 mg, 97%). Thefiltrate was evaporated to give a residue which was redissolved inacetic acid. Following lyophilization the acetate salt was obtained as awhite solid (83.9 mg, 61%).

To a solution of 17-tetrabenzo(a,c,g,i)fluorenylmethyl-para-nitrophenylcarbonate (139.4 mg, 0.248 mmol) and L-phenylalanine tert-butyl esteracetate (83.9 mg, 0.298 mmol; 1.2 equiv) in dichloromethane (5 ml) wasadded N,N′-dimethylaniline (63 μl, 0.497 mmol; 2 equiv). The reactionmixture was stirred at room temperature under nitrogen for 120 h. Afteraddition of water (10 ml) and acidification with KHSO₄ (2M) to pH=1, thereaction mixture was extracted with dichloromethane (3×20 ml). Thecombined organic phases were washed with water (2×15 ml) to pH 6-7 anddried over MgSO₄. After filtration the solvent was evaporated underreduced pressure to give an orange oil. After purification by flashchromatography using toluene as the eluent, the fractions containingmaterial of R_(f)=0.04 were evaporated to give a yellow solid.Recrystallization from ether/petrol (b.p. 40-60° C.) gave compound (54)as a pale yellow solid which was filtered, washed with petrol andfinally dried (73.9 mg, 46%);

m.p. 158-162° C. (dec); t.l.c. R_(f)(C) 0.04, R_(f)(H) 0.84; ν_(max)(KBr) 3280 (NH stretching), 3060, 3030 (CH stretching, aryl), 2980, 2930(CH stretching, alkyl), 1735 (CO, ester), 1715 (CO, urethane), 1680(amide I), 1610 (aromatic rings), 1545 (amide II), 1500 (aromaticrings.) 1440 (CH deformations, alkyl), 1390, 1370 (CH₃, symmetricaldeformations), 1290, 1255, 1220, 1155, 1050 (CO stretching), 850, 750,720, 700 (out-of-plane CHdeformation) cm⁻¹; m/z (FAB) 643 (M⁺), 379.HRMS 643.27225, C₄₄H₃₇NO₄ requires: 643.27224 ( ) <1 ppm.

Nα-17-Tetrabenzo(a,c,g,i)fluorenylmethoxycarbonyl-L-phenylalanine(Tbfmoc-L-Phe OH) (55)

A solution ofNα-17-tetrabenzo(a,c,g,i)fluorenylmethoxycarbonyl-L-phenylalaninetert-butyl ester (68.2 mg, 0.106 mmol) in trifluoroacetic acid (950 μl)and water (50 μl) was sonicated for 2.5 h. The solvent was removed invacuo to give a purple residue. A mixture of diethyl ether and petrolb.p. 40-60° C. (1:1) was added. The precipitate obtained was chilledovernight, filtered, washed with petrol and finally dried in a vacuumdesiccator over P₂O₅ to give compound (55) (57.6 mg, 93);

m.p. 137-140° C.; [d]D −50.8 (C=0.25, CH₂Cl₂); t.l.c.: R^(f)(H) 0.43,R_(f)(I) 0.89; ν_(max) (KBr) 3410 (NH stretching), 3060, 3030 (CHstretching, aryl), 2960, 2860 (CH stretching, alkyl), 1715 (CO, acid andurethane), 1610, 1500 (aromatic rings), 1435, 1420 (CH deformations,alkyl), 1340 (OH bending), 1215, 1050 (CO stretching), 750, 730, 700(out-of-plane CH deformation) cm⁻¹; ν_(max) 384 (15794), 368 (16896),302 (39670), 290 (32324), 262 (59505), 254 (64647) nm; δH (CDCl₃, 200MHz) 8.80-8.58 (6H, m, aromatic), 8.30-8.19 (2H, m, aromatic), 7.65-7.54(8H, m, aromatic), 7.19-7.07 (5H, m, aromatic (Phe)), 5.13 (1H, apparentt, CH), 4.98 (1H, d, ₃J=8.4 Hz, NH), 4.79-4.64 (2H, m, H_(a) (CH₂) andαCH (Phe)), 4.30-4.24 (1H, m, H_(b) (CH₂)), 3.08 (2H, m, β CH₂ (Phe));δc (CDCl₃, 90 MHz), 175.8 (CO, acid), 155.8 (CO, urethane), 142.6,141.0, 136.8, 136.5, 135.3, 131.5, 130.3, 130.1 (quaternary aromaticC's), 129.0, 128.6, 127.4, 127.3, 127.1, 126.8, 126.6, 125.9, 125.7,125.5, 125.1, 124.9, 124.8, 123.4, 123.1, 123.0 (aromatic CH's), 69.2(CH₂), 54.4 (α CH), 47.4 (CH), 37.4 (β CH₂); m/z (FAB) 587 (M⁺), 379.HRMS 588.21744, C₄₀H₃₀NO₄ requires: 588.21747 ( ) <1 ppm.

7 6 amino acids amino acid linear peptide 1 Gly Ser Met Val Leu Ser 5 5amino acids amino acid linear peptide 2 Ser Met Val Leu Ser 1 5 24 aminoacids amino acid linear peptide 3 Gly Arg Thr Leu Ser Asp Tyr Asn IleGln Lys Glu Ser Thr Leu His 1 5 10 15 Leu Val Leu Arg Leu Arg Gly Gly 2023 amino acids amino acid linear peptide 4 Arg Thr Leu Ser Asp Tyr AsnIle Gln Lys Glu Ser Thr Leu His Leu 1 5 10 15 Val Leu Arg Leu Arg GlyGly 20 42 amino acids amino acid linear peptide 5 Gly Ile Pro Pro AspGln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu 1 5 10 15 Glu Asp Gly ArgThr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr 20 25 30 Leu His Leu ValLeu Arg Leu Arg Gly Gly 35 40 41 amino acids amino acid linear peptide 6Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu 1 5 1015 Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu 20 2530 His Leu Val Leu Arg Leu Arg Gly Gly 35 40 40 amino acids amino acidlinear peptide 7 Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln LeuGlu Asp 1 5 10 15 Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu SerThr Leu His 20 25 30 Leu Val Leu Arg Leu Arg Gly Gly 35 40

What is claimed is:
 1. A process for synthesis or separation of amixture of compounds, comprising the steps of: a) protecting at leastone group in at least one compound in a mixture of compounds to beseparated with a protective group having the formula (I): Ar—L—  (I) wherein Ar represents a substantially planar fused ring systemcontaining at least 4 aromatic rings; and L represents a groupcontaining at least one carbon atom which is capable of bonding to agroup to be protected; and b) passing the mixture of compounds through achamber filled with a graphite material.
 2. A process for synthesis orseparation of a mixture of compounds, comprising the steps of: a)protecting at least one group in at least one compound selected frompeptide, a nucleoside and a nucleotide, in a mixture of compounds to beseparated, with a protective group having the formula (I):  Ar—L  (I) wherein Ar represents a substantially planar fused ring systemcontaining at least 4 aromatic rings; and L represents a groupcontaining at least one carbon atom which is capable of bonding to agroup to be protected; and b) passing the mixture of compounds through achamber filled with a graphite material.
 3. A process for synthesis orseparation of a mixture of compounds, comprising the steps of: a)protecting at least one group in at least one compound in a mixture ofcompounds to be separated with a protective group having the formula(I): Ar—L  (I)  wherein Ar represents a substantially planar fused ringsystem containing at least 4 aromatic rings; and L represents a groupcontaining at least one carbon atom which is capable of bonding to agroup to be protected; b) passing the mixture of compounds through achamber filled with a graphite material; and c) removing the protectivegroup from said at least one group in said at least one compound.
 4. Aprocess according to claim 3 wherein the protective group has theformula (IA): Ar—(CH₂)_(n)—CRR′—  (IA) wherein R and R′ are eachhydrogen, alkyl, aryl, aralkyl or cycloalkyl; and n is an integer offrom 0 to 5, and is removed by acidolysis.
 5. A process according toclaim 3 wherein the protective group has the formula (IB) or (IC):Ar—(CH₂)_(n)—C(CY_(m))(R)—  (IB) Ar—(CH₂)_(n)—(CF₂)_(m)—C(R)₂—  (IC)wherein R is hydrogen, alkyl, aryl, aralkyl or cycloalkyl; Y is halogen;n is an integer of from 0 to 5; and m is an integer of from 1 to 8, andis removed by treatment with zinc and acetic acid.
 6. A processaccording to claim 3 wherein the protective group has the formula (ID):Ar—(CH₂)_(n)—CH═C(R)—CHR′—  (ID) wherein R and R′ are each hydrogen,alkyl, aryl, aralkyl or cycloalkyl; and n is an integer of from 0 to 5,and is removed by treating said at least one group in at least onecompound with a catalyst comprising a Group VIII element complexed witha water-soluble coordinating agent in an aqueous phase.
 7. A processaccording to claim 3 wherein the protective group has the formula (ID):Ar—(CH₂)_(n)—CH═C(R)—CHR′—  (ID) wherein R and R′ each hydrogen, alkyl,aryl, aralkyl or cycloalkyl; and n is an integer of from 0 from to 5,and is removed by hydrogenation over a hydrogenation catalyst.
 8. Aprocess for synthesis or separation of a mixture of compounds,comprising the steps of: a) protecting at least one group in at leastone compound in a mixture of compounds to be separated with a protectivegroup having the formula (I): Ar—L  (I)  wherein Ar represents asubstantially planar fused ring system containing at least 6 aromaticrings; and L represents a group containing at least one carbon atomwhich is capable of bonding to a group to be protected; and b) passingthe mixture of compounds through a chamber filled with a graphitematerial.
 9. A process according to claim 8 wherein the aromatic ringsare hexagonal.
 10. A process for synthesis or separation of a mixture ofcompounds, comprising the steps of: a) protecting at least one group inat least one compound in a mixture of compounds to be separated with aprotective group having the formula (I):  Ar—L  (I)  wherein Arrepresents a substantially planar fused ring system containing at least4 hexagonal aromatic rings; and L represents a group containing at leastone carbon atom which is capable of bonding to a group to be protected;and b) passing the mixture of compounds through a chamber filled with agraphite material.
 11. A process for synthesis or separation of amixture of compounds, comprising the steps of: a) protecting at leastone group in at least one compound in a mixture of compounds to beseparated with a protective group having the formula (I): Ar—L  (I) wherein Ar represents a substantially planar fused ring systemcontaining at least 4 aromatic rings and wherein Ar contains noheteroatom; and L represents a group containing at least one carbon atomwhich is capable of bonding to a group to be protected; and b) passingthe mixture of compounds through a chamber filled with a graphitematerial.
 12. A process for synthesis or separation of a mixture ofcompounds, comprising the steps of: a) protecting at least one group inat least one compound in a mixture of compounds to be separated with aprotective group having the formula (II):

 wherein the protective group is a substantially planar fused ringsystem containing at least 4 aromatic rings; n is an integer of 0 or 1;O means that the surrounding ring is aromatic when n is 1 andnon-aromatic when n is 0; R₁ and R₂ together and R₃ and R₄ together forma fused aromatic ring system together with the carbon atoms to whichthey are attached; R and R′ are each an alkyl group or hydrogen,provided that when n is 1, R′ is absent; and L′ represents a direct bondor a group capable of bonding to a group to be protected; and b) passingthe mixture of compounds through a chamber filled with a graphitematerial.
 13. A process according to claim 12 wherein L′ is —CO—,—O—CO—, —S—, —O—, —(CH₂)_(m)—O—, wherein m is from 1 to 6, or a directbond.
 14. A process for synthesis or separation of a mixture ofcompounds, comprising the steps of: a) protecting at least one group inat least one compound in a mixture of compounds to be separated with aprotective group having the formula (III):

 wherein n is an integer of 0 or 1; O means that the surrounding ring isaromatic when n is 1 and non-aromatic when n is 0; R₁ and R₂ togetherand R₃ and R₄ together form a fused aromatic ring system together withthe carbon atoms to which they are attached; R and R′ are each an alkylgroup or hydrogen, provided that when n is 1, R′ is absent; L′represents a direct bond or a group capable of bonding to a group to beprotected; R₅ is a group which, together with the atoms to which it isattached, forms one or more supplementary rings; and X represents a bondto R₅ or a hydrogen atom; and b) passing the mixture of compoundsthrough a chamber filled with a graphite material.
 15. A processaccording to claim 14 wherein L′ is —CO—, —O—CO—, —S—, —O—,—(CH₂)_(m)—O—, wherein m is from 1 to 6, or a direct bond.